CN116966162A

CN116966162A - Immunomodifying particles for treating inflammation

Info

Publication number: CN116966162A
Application number: CN202310533083.XA
Authority: CN
Inventors: N·金; D·格茨
Original assignee: Oncour Pharma Inc
Current assignee: Oncour Pharma Inc
Priority date: 2013-03-13
Filing date: 2014-03-13
Publication date: 2023-10-31
Also published as: EP3494966A1; JP2022184935A; WO2014160465A3; AU2014243758A1; US20150010631A1; AU2021221379B2; AU2023263476A1; JP2019073550A; JP6725413B2; MX2021005471A; US11045492B2; IL303425A; KR20230025948A; JP2020172501A; IL292823B1; HK1220368A1; CN105263476A; IL241553B; AU2014243758B2; EP2968160A4

Abstract

The present application relates to the administration of negatively charged particles such as polystyrene, PLGA or diamond particles to a subject to improve inflammatory immune responses. In addition, methods of treating inflammatory diseases by administering these same negatively charged particles are described.

Description

Immunomodifying particles for treating inflammation

The application is a divisional application of Chinese patent application with the application number of 202111245767.7 and the name of 'immune modification particles for treating inflammation', which is filed on the application day of 2014, 3 and 13.

The application is a divisional application of Chinese patent application with the application number of 201480026034.6 and the name of 'immune modification particles for treating inflammation', which is filed on the application day of 2014, 3 and 13.

Cross Reference to Related Applications

The present application calls for U.S. provisional application No.61/779,182 filed on day 13 of 3.2013; U.S. provisional application No.61/844,961 filed on 7.11.2013; priority is given to U.S. provisional application No.61/865,392 filed on day 13 of 8, 2013 and U.S. provisional application No.61/877,212 filed on day 4 of 10, 2013, each of which is hereby incorporated by reference in its entirety.

Description of electronically submitted text files

The contents of the text file submitted electronically therewith are incorporated herein by reference in their entirety: a computer readable format copy of the sequence listing (file name: COUR-001_01wo_st25.Txt, date of record: 13 days 3 in 2014, file size 1.2 megabytes).

Background

Inflammatory diseases and disorders are conditions in which an abnormal or otherwise deregulated inflammatory response contributes to the etiology or severity of the disease. Examples include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, celiac disease and diabetes; infectious diseases such as tuberculosis and various forms of meningitis and encephalitis, including west nile virus encephalitis; and ischemia reperfusion diseases such as myocardial infarction and graft reperfusion injury. In addition, exacerbations of inflammation observed after surgery, injury, or other tissue trauma can have an adverse effect on patient recovery.

Many of these diseases or disorders are characterized by multi-core/mononuclear cell infiltration at the site of tissue injury or other damage. Examples of monocytes that have been observed in these infiltrates include lymphocytes, especially T lymphocytes, and cells of the mononuclear phagocyte system (MPS cells), such as monocytes, macrophages, dendritic cells, microglia and other cells.

Many of the cells observed in mononuclear cell infiltration are suspected to have a role in these aberrant inflammatory responses. For example, in diseases such as multiple sclerosis, CD4 is known ⁺ T cells play a central role in the pathological autoimmune response. At an earlier time point in T cell activation, dendritic cells and other MPS cells may be responsible for CD4 ⁺ Activation of T cells. MPS cells can also promote inflammation by phagocytosis, but in at least some inflammatory diseases, it is not clear that CD4 is absent ⁺ In the case of T cells, whether such cells are capable of doing so.

Peripheral blood mononuclear cells can be classified into one or more groups based on whether certain cell surface molecules are expressed or not. In particular, human "resident monocytes" or "mature monocytes" are understood to have CD14 ^lo CD16 ⁺ Phenotype (mouse corresponding phenotype is CX) ₃ CR1 ^hi CCR2 ^- Gr1 ^- ). Another group of cells "inflammatory monocytes" or "immature monocytes" is understood to have CD14 ⁺ CD 16-phenotype (mouse corresponding phenotype is CX ₃ CR1 ^lo CCR2 ⁺ Gr1 ⁺ ). (Geissmann F.et al 2003Immunity 19:71-82 (Geissmann F.et al, 2003, immunity, volume 19, pages 71-82))

Inflammatory monocytes have been found to play a role in a wide range of immune-mediated diseases and disorders. LY6C after entering inflamed tissue ^hi Inflammatory monocytes differentiate into tissue macrophages or Dendritic Cells (DCs), which secrete a variety of pro-inflammatory cytokines, proteases, and other mediators, including nitric oxide, ultimately leading to tissue damage, scarring, and even death. (Getts et al20098 j.exp.med.205:2319-2337 (Getts et al, 2008, journal of experimental medicine, volume 205, pages 2319-2337); lin et al,2011J Immunol 186:508-515 (Lin et al,2011, journal of immunology, volume 186, pages 508-515); schiopu et al 2012 Atheroscleosis 223:291-298 (Schiopu et al 2012, atherosclerosis, volume 223, pages 291-298); swirski et al 2010J Clin Invest 120:2627-2634 (Swirski et al, 2010, journal of clinical research, volume 120, pages 2627-2634); swirski et al 2009Science 325:612-616 (Swirski et al 2009, science, volume 325, pages 612-616); getts et al, j. Neuroisflamm. In press (Getts et al, journal of neuroinflammation, publications); nahrendorf et al 2010Circulation 121:2437-2445 (Nahrendorf et al, 2010, cycle 121, pages 2437-2445)). Inhibition of inflammatory monocytes not only reduces pathological states, but also is able to initiate early repair mechanisms in certain inflammatory disorders. To date, no safe and effective therapies have been able to specifically target inflammatory monocytes to alter these prognosis. Physicians rely on widely acting steroidal, non-steroidal anti-inflammatory agents or antibodies that transiently neutralize the components of the inflammatory monocyte response.

Given the role of inflammatory monocytes and polymorphonuclear cells in most disease conditions, their therapeutic accessibility in the blood stream, and their inherent propensity to interact with particles, particle-based therapeutics may target these cells more conditionally specific than antibodies or small molecules. To date, work in this area has focused on the formulation of slow-release small molecule therapeutics conjugated to nanoparticles or microparticles to be able to enhance the delivery of cancer therapeutics or antigen materials for vaccination purposes (De Jong et al 2008Int J Nanomedicine 3:133-149 (De Jong et al 2008, journal of nanomedicine international, volume 3, pages 133-149)). However, the in vivo immunomodulatory potential of the particles themselves, which do not carry the active pharmaceutical ingredient, is in most cases neglected.

The ability of natural leukocytes to clear and induce apoptosis remains a major goal of therapies aimed at alleviating pathological conditions associated with specific cell subsets, including inflammatory-derived macrophages and dendritic cells. It has surprisingly been found that Immune Modified Particles (IMP) derived from polystyrene, nanodiamond or biodegradable poly (lactic-co-glycolic acid) are taken up by inflammatory monocytes via collagen-like structure macrophage receptors (MARCO) upon infusion, triggering monocyte migration and entrapment in the spleen, where they undergo caspase 3 mediated apoptosis. It may be even more surprising that targeted administration of Immune Modifying Particles (IMP) in acute models of inflammation not only reduces inflammatory monocyte accumulation at the site of primary inflammation, but also reduces the pathological state and disease severity in all models. IMP is a general, easy-to-transform therapeutic option for diseases caused or enhanced by inflammatory monocytes. IMP represents a novel and safe therapy specific for inflammatory monocytes.

Regulatory T cells (or tregs) are the type of immunoregulatory cells in the periphery that are important in controlling autoimmune and systemic inflammation. Tregs are CD4 positive cells that normally constitutively express CD 25. Thus, tregs are typically CD4 ⁺ CD25 ⁺ T cells. These regulatory T cells are potent inhibitors of T cell mediated immunity in a range of inflammatory disorders including, but not limited to, infectious diseases, autoimmunity, pregnancy, and tumors. In vivo, small amounts of tregs can control a large number of activated effector T cells. While freshly isolated tregs exhibit minimal constitutive inhibitory function, connecting T cell antigen receptor (TCR) in vitro or pre-immunizing mice with high doses of autoantigens in vivo stimulates Treg inhibitory function. TCR ligation is abnormal in the necessity to enhance T cell inhibitory function, as most T cells are thought to recognize constitutively expressed autoantigens.

It is desirable to enhance Treg suppression function in inflammatory conditions such as autoimmune diseases in order to alleviate inflammatory responses to self-proteins. Conversely, for example, when attempting to enhance an immune response to a tumor, it may be desirable to turn off Treg function by blocking Treg function or reprogramming Treg into effector T cells capable of producing an inflammatory immune response to the desired target.

Disclosure of Invention

The present invention relates to the surprising discovery that: the modified particles alone (i.e., without the peptide coupled thereto) are effective in ameliorating an inflammatory immune response in a patient in need thereof. Surprisingly, all that is required to inhibit the inflammatory immune response and treat inflammatory diseases is to administer negatively charged particles without having to couple peptides thereto.

In one embodiment, the invention provides a method of inducing apoptosis of monocytes, granulocytes and/or neutrophils in a subject comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In other embodiments, the negatively charged particles are poly (lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles areStabilized polypropylene polysulfide particles. In other embodiments, the negatively charged particles are carboxylated.

In one embodiment, the invention provides a method of inducing and expanding anti-inflammatory cd103+ dendritic cells and regulatory T cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety.

In one embodiment, the invention provides a method of removing a pro-inflammatory mediator from the inflammatory environment of a subject having an inflammatory disorder, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In other embodiments, the negatively charged particles are poly (lactic-co-glycolic acid) (PLGA) particles. In one placeIn some embodiments, the particles areStabilized polypropylene polysulfide particles. In another embodiment, the negatively charged particles are carboxylated. In another embodiment, the negatively charged particles bind to a pro-inflammatory polypeptide produced in the subject.

In one embodiment, the invention provides a method of concentrating and presenting a regulatory protein from the inflammatory environment of a subject having an inflammatory disorder, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In other embodiments, the negatively charged particles are poly (lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are Stabilized polypropylene polysulfide particles. In another embodiment, the negatively charged particles are carboxylated. In another embodiment, the negatively charged particles bind to regulatory proteins produced in the subject.

In yet another embodiment, the invention provides a method for controlling a pathological and/or unwanted inflammatory immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles that are pre-absorbed with a regulatory protein such that, after administration, the negatively charged particles concentrate and present the regulatory protein to ameliorate the inflammatory immune response in the subject. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In other embodiments, the negatively charged particles are poly (lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles areStabilized polypropylene polysulfide particles. In another embodiment, the negatively charged particles are carboxylated.

In one embodiment, the invention provides a method of inducing antigen-specific tolerance in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles having one or more antigens embedded therein, and a carrier. In another embodiment, the negatively charged particles do not contain an attached peptide or antigen moiety. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In other embodiments, the negatively charged particles are poly (lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are Stabilized polypropylene polysulfide particles. In another embodiment, the negatively charged particles are carboxylated. In another embodiment, the negatively charged particles bind to a pro-inflammatory polypeptide produced in the subject.

In one embodiment, the particles are coupled to an antigen comprising one or more epitopes. In another embodiment, the epitope is associated with an allergic reaction, an autoimmune disease, or an inflammatory disease or disorder. In one embodiment, the epitope is associated with type 1 diabetes, multiple sclerosis, celiac disease, or inflammatory bowel disease (including crohn's disease or colitis, e.g., ulcerative colitis). In one embodiment, the epitope is an epitope as described in table 1 or 2. In one embodiment, the particles are coupled to an antigen comprising only one epitope associated with one disease and/or disorder. In another embodiment, the antigen comprises more than one epitope associated with the same disease and/or disorder. In another embodiment, the antigen comprises more than one epitope associated with different diseases and/or disorders.

In one embodiment, the negatively charged particles have a zeta potential of less than about-100 mV. In another embodiment, the negatively charged particles have a zeta potential of less than about-50 mV. In one embodiment, the negatively charged particles have a zeta potential of between-100 mV and 0 mV. In some embodiments, negatively charged particles having a zeta potential of between-75 mV and 0mV are carboxylated. In another embodiment, the negatively charged particles have a zeta potential of between-60 mV and 0 mV. In another embodiment, the negatively charged particles have a zeta potential of between-50 mV and 0 mV. In a specific embodiment, the negatively charged particles have a zeta potential of between-50 mV and-40 mV. In another embodiment, the charged particles have a zeta potential of between-100 mV and-50 mV. In one embodiment, the charged particles have a zeta potential of between-75 mV and-50 mV.

In one embodiment, the pharmaceutical formulation of the invention reduces and/or inhibits infiltration of inflammatory lesions by inflammatory monocytes. In another embodiment, the pharmaceutical formulation of the invention improves inflammatory immune response.

In one embodiment, the pharmaceutical formulation of the invention increases the number of regulatory T cells. In another embodiment, the pharmaceutical formulation of the invention improves inflammatory immune response.

In one embodiment, the pharmaceutical formulation of the present invention comprises negatively charged particles having an average diameter of about 0.1 μm to about 10 μm. In another embodiment, the negatively charged particles have an average diameter of about 0.2 μm to about 2 μm. In another embodiment, the negatively charged particles have an average diameter of about 0.3 μm to about 5 μm. In yet another embodiment, the negatively charged particles have an average diameter of about 0.5 μm to about 3 μm. In yet another embodiment, the negatively charged particles have an average diameter of about 0.5 μm.

In one embodiment, the subject has received or will receive surgery. In another embodiment, the negatively charged particles are administered to the subject prior to surgery. In yet another embodiment, negatively charged particles are administered to a subject during surgery. In yet another embodiment, the negatively charged particles are administered to the subject after surgery. In another embodiment, the subject is a transplant recipient.

In another embodiment, the subject has recently experienced physical trauma. In another embodiment, the subject has recently experienced an injury. In yet another embodiment, the injury is a sports injury. In another embodiment, the injury is concussion.

In one embodiment, the subject has an autoimmune disorder. In another embodiment, the autoimmune disorder is multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, or crohn's disease. In a specific embodiment, the autoimmune disease is multiple sclerosis. In another embodiment, the autoimmune disease is celiac disease. In another embodiment, the autoimmune disease is type 1 diabetes. In another embodiment, the autoimmune disease is an inflammatory bowel disease, including crohn's disease and ulcerative colitis.

In one embodiment, the subject has an allergic disorder. In another embodiment, the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity. In another embodiment, the subject is a transplant recipient.

In one embodiment, the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction. In another embodiment, the subject has psoriasis or dermatitis.

In one embodiment, the subject has a viral infection. In another embodiment, the viral infection is a herpes virus infection, a hepatitis virus infection, a west nile virus infection, a flavivirus, an influenza virus infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection. In another embodiment, the viral infection causes the central nervous system of the subject to be infected. In yet another embodiment, the viral infection causes viral encephalitis or viral meningitis.

In one embodiment, the subject has a bacterial infection. In another embodiment, the bacterial infection infects the central nervous system of said subject. In yet another embodiment, the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

In another embodiment, administration of the particles of the invention prevents accumulation of neutrophils and other granulocytes in the subject that may cause a pathological condition. In another embodiment, the subject has cancer.

In one embodiment, administration of the particles of the invention enhances regeneration of damaged tissue in a subject. In one embodiment, administration of the particles of the invention enhances regeneration of damaged tissue in a subject with Amyotrophic Lateral Sclerosis (ALS) or post-traumatic stress disorder (PTSD). In another embodiment, administration of the particles enhances regeneration of epithelial cells. In yet another embodiment, administration of the particles enhances remyelination of neurons. In another embodiment, the subject has an autoimmune disease. In yet another embodiment, the subject has inflammatory bowel disease, ulcerative colitis, and/or crohn's disease. In yet another embodiment, the subject has multiple sclerosis. In one embodiment, administration of the negatively charged particles induces antigen-specific tolerance in the subject. In one embodiment, the antigen-specific tolerance-inducing particles comprise one or more epitopes associated with allergies, autoimmune diseases and/or inflammatory diseases. In one embodiment, the epitope is selected from those described in table 1 or 2. In one embodiment, the negatively charged particles are polystyrene, diamond,Stabilized polypropylene sulfide or poly (lactic-co-glycolic acid) particles. In one embodiment, the particles are carboxylated. In one embodiment, the particles have a zeta potential of less than about-100 mV. In one embodiment, the particles have a zeta potential of between about-75 mV and 0mV, for example, between-50 mV and 0mV, or between-100 mV and-50 mV, or between-75 mV and-50 mV, or between-50 mV and-40 mV. In one embodiment, the particles have an average diameter of about 0.1 μm to about 10 μm, such as about 0.2 μm to about 2 μm, or about 0.3 μm to about 5 μm, or 0.5 μm to about 3 μm, or about 0.5 μm to about 1 μm.

In one embodiment, the subject has an autoimmune disease. In one embodiment, the autoimmune disease is multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, or crohn's disease. In one embodiment, the particles comprise one or more myelin basic protein epitopes. In one embodiment, the myelin basic protein epitope is one or more of SEQ ID NO. 4975 or SEQ ID NO. 4976. In one embodiment, the particles comprise one or more myelin oligodendrocyte glycoprotein epitopes. In one embodiment, the myelin oligodendrocyte glycoprotein epitope is one or more of SEQ ID NO:1 or SEQ ID NO: 4978. In one embodiment, the particles comprise one or more insulin epitopes. In one embodiment, the one or more insulin epitopes are SEQ ID NO 4981. In one embodiment, the particle comprises one or more glutamate decarboxylase epitopes. In one embodiment, the glutamate decarboxylase epitope is SEQ ID NO:4982. In one embodiment, the particle comprises one or more proteolipid protein epitopes. In one embodiment, the proteolipid protein epitope is SEQ ID NO. 4977. In one embodiment, the particle comprises one or more gliadin epitopes. In one embodiment, the gliadin epitope comprises SEQ ID NO:4983-4985.

In one embodiment, the method comprises applying negatively charged particles by any suitable means. In one embodiment, the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously. In particular embodiments, carboxylated particles are administered nasally. In yet another embodiment, the negatively charged particles are administered intravenously. In yet another embodiment, the negatively charged particles are administered subcutaneously.

In one embodiment, the method comprises a method of diagnosing an inflammatory disorder in a subject. In particular embodiments, the method comprises removing blood from a subject and incubating the blood or serum/plasma derived therefrom with negatively charged particles, and determining whether the proteins listed in table 6 are present, wherein the presence of one or more proteins of table 6 is indicative of an inflammatory immune response. In another embodiment, the method comprises injecting a pharmaceutical composition comprising negatively charged particles into the blood circulation of a subject, and subsequently removing the particles from the blood circulation, and determining whether the proteins listed in table 6 are present on the surface of the particles removed from the subject, wherein the presence of one or more proteins of table 6 is indicative of an inflammatory immune response.

Drawings

FIG. 1 shows the ability of (A-G) treating WNV infected mice with PS-IMP to ameliorate disease pathology. WNV infected mice were treated with PS-IMP at > 5% weight loss, so that mice that would otherwise die from the disease achieved 60% survival (a). This effect was significantly reduced in mice treated with polystyrene neutral particles (PSNP) and was absent in vehicle controls (a). On day 7 post immunization, infiltration of inflammatory monocyte-derived macrophages into the brain of WNV infected mice was significantly reduced in mice treated with polystyrene immune-modified particles (PS-IMP) and to a lesser extent with NP treated mice (b). Treatment of WNV infected animals with poly (lactic-co-glycolic acid) immunomodified particles (PLGA-IMP) or Nanodiamond (ND) -IMP also resulted in about 60% survival of mice that would otherwise die from infection (c). The negative charge of IMP is critical for this effect, since treatment of WNV infected mice with polystyrene based polystyrene positive particles (PS-PP) did not reduce inflammatory monocyte macrophage infiltration into the brain at day 7 post immunization (d) or improve survival (e). The optimal dose of PS-IMP to promote survival in WNV infected mice has been determined to be 0.355mg, corresponding to about 4X 109 particles (f, g). Survival data represent three separate experiments with 10-20 mice/group. Statistical analysis was performed using the Mantel-Haenszel log rank test. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Statistical analysis was performed using one-way anova and Tukey-Kramer post hoc test. When comparing PS-IMP group and NP group with vehicle control group, P was less than or equal to 0.05 (.x), P was less than or equal to 0.01 (.x), and P was less than or equal to 0.001 (.x). When comparing PS-IMP group with NP group, P.ltoreq.0.05 (#), P.ltoreq.0.01 (#), P.ltoreq.0.001 (# #).

Fig. 2 shows (a-G) IM redirection to the spleen when treated with PS-IMP. Twenty-four hours after intravenous injection, FITC-PS-IMP localizes to the lung, spleen and liver, whereas it is rarely observed in the brain, kidneys or thymus (a). FITC-PS-IMP in the spleen was similar to Ly6C in amounts in mock-infected animals ^hi ΦIM、B220 ⁺ B cell, CD3 ⁺ T cells and NK1.1 ⁺ NK cell correlation (b, c). In WNV infected animals, FITC-PS-IMP is predominantly localized to Ly6C ^hi Φim (b, c). (d) Spleens were treated for flow cytometry at day 7 post immunization and gating on living cells (R1). From this population, CD45 was selected ⁺ White blood cells (R2), and on CD11b ⁺ Ly 6G-monocytes are gated to exclude Ly6G from a small population ⁺ Neutrophils (R3; d). FITC-PS-IMP was found to be associated with Ly6C in WNV infected animals ^hi Phi IM related, in contrast to Ly6C in mock infected animals ^lo Monocyte related (e). On day 7 post immunization of WNV infected animals, infusion of PS-IMP and to a lesser extent NP resulted in Ly6C ^hi Phi IM accumulates in the spleen (f) and the number of these cells is significantly reduced in the blood (g). Immunohistochemical data represents three separate experiments with 3 mice/group. Slides were counterstained with DAPI (blue) to identify nuclei. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Statistical analysis was performed using one-way anova and Tukey-Kramer post hoc test. When comparing PS-IMP group and NP group with vehicle control group, P was less than or equal to 0.05 (.x), P was less than or equal to 0.01 (.x), and P was less than or equal to 0.001 (.x). When comparing PS-IMP group with NP group, P is less than or equal to 0.05% ^# )，P≤0.01( ^## )，P≤0.001( ^### )。

Figure 3 shows that (a-F) PS-IMP treatment reduced homing of inflammatory monocytes to the CNS of WNV infected mice. Ly6C was isolated from bone marrow of donors mimicking infection and WNV infection at day 6 post immunization ^hi /CD11b ⁺ /CD11c ^- /Ly6G ^- Phi IM, on day 6 post immunization, was labeled with PKH26 and injected into matched recipients, followed by separate injections of vehicle, NP or PS-IMPa) A. The invention relates to a method for producing a fibre-reinforced plastic composite Treatment of brain (b) and spleen (c) for flow cytometry at day 7 post immunization, and for CD45 ^hi /CD11b ⁺ Macrophages are gated (R1). PS-IMP significantly reduces adoptive transfer of PKH26 ⁺ /Ly6C ^hi Infiltration of bone marrow derived Φim into brain (b, d), which is compatible with PKH26 ⁺ The accumulation of cells in the spleen is related (c, e). It was found that more than 70% of the adoptive transferred Φim ingested FITC-PS-IMP (i.e., PKH26 ⁺ /FITC ⁺ A cell; g, R1), cells expressing CD11c and CD103 (g, R2). Infusion of PS-IMP into splenic mice at 5% weight loss (f) failed to reduce the transport of monocytes into the brain, with PS-IMP treated and vehicle treated animals having similar numbers of monocytes as determined by flow cytometry (f). Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Statistical analysis was performed using one-way anova and Tukey-Kramer post hoc test. When comparing PS-IMP group and NP group with vehicle control group, P was less than or equal to 0.05 (.x), P was less than or equal to 0.01 (.x), and P was less than or equal to 0.001 (.x). When comparing PS-IMP group with NP group, P is less than or equal to 0.05% ^# )，P≤0.01( ^## )，P≤0.001( ^### )。

FIG. 4 shows that (A-I) inflammatory monocytes express MARCO that is critical for IMP activity. The ability of PS-IMP to inhibit the migration of monocytes into thioglycolate inflammatory peritoneum was tested (protocol shown in a). Wild type and MARCO ^-/- Mice respond similarly to thioglycolate injection, e.g. by Ly6C isolated from the peritoneum ^hi /CD11b ⁺ The number of monocytes (ΦIM) was determined (b, e). PS-IMP treatment at 48 hours (as shown in a) significantly reduced infiltration of Φim into the peritoneal cavity, but not from MARCO, in wild-type animals injected with thioglycolate ^-/- Φim isolated from peritoneal cavity of mice (b, e). It was found that FITC-PS-IMP (green) and MARCO in the spleen border region ⁺ (red fluorescent) cell-associated (c). WNV infection upregulates MARCO expression on Φim (d). In wild-type mice injected with thioglycolate, PS-IMP treatment correlated with a significant increase in the number of Φim in the spleen, in MARCO ^-/- No such was observed in the animals (b),e) A. The invention relates to a method for producing a fibre-reinforced plastic composite With MARCO ^-/- Isolation of significantly higher amounts of PS-IMP from spleens of wild-type mice injected with thioglycolate compared to animals ⁺ Ly6C ^hi Φim (f). Infusion of PS-IMP into thioglycolate-induced wild-type mice resulted in Ly6C expressing the apoptosis markers annexin V (g, h) and caspase-3 (i) ^hi The number of phiims increases significantly. In MARCO ^-/- This was not observed in animals. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Statistical analysis was performed using one-way anova and Tukey-Kramer post hoc test. When comparing the wild-type PS-IMP group with all other groups, p.ltoreq.0.05 (.

FIG. 5 shows that (A-S) PS-IMP improves the disease pathology and inflammatory mononuclear cell infiltration in EAE, myocardial inflammation and inflammatory bowel disease. In use of PLP _139-151 In immunized SJL mice, PLGA-IMP infusion initiated at EAE onset (a) or initial relapse (b) significantly reduced the mean clinical score. Flow cytometry of spinal cord from mice treated with PLGA-IMP or vehicle for 7 days post-autoimmunization showed significantly less inflammation, as evidenced by reduced CD45 ⁺ CD11b ⁺ CD11c ⁺ Ly6C ^hi Cell assay (c). This reduced monocyte influx into the spinal cord and increased CD45 in the spleen ⁺ CD11b ⁺ Ly6C ⁺ Monocyte related (d). PLGA-IMP was also tested as another model of inflammation in the permanent left anterior descending branch arterial occlusion model (infusion as shown in h). PLGA-IMP infusion for three days as shown in (H) resulted in a reduction in infarct size compared to vehicle-treated controls, H as described in "materials and methods &E histological and image analysis (i, j). In addition, the reduced occlusion size and less CD68 in PLGA-IMP treated mice compared to vehicle treated controls ⁺ Macrophage associated (k, l, red fluorescence). In another model of inflammation, the artery is ligated for 45 minutes, after which the blood flow is returned. Mice were treated with PLGA-IMP for 6 days as indicated in (m). On days 1 and 5, the IMP treatment was performed as compared to vehicle-treated controlsSerum creatinine clearance is significantly greater in animals of (n). This is associated with reduced tubular atrophy in IMP treated animals (o). As another model of inflammation, PS-IMP therapy was tested in a DSS-induced colitis model using the PS-IMP or vehicle infusion regimen shown in (p), alleviating the overall clinical severity (q) of DSS-induced colitis. Reduced clinical score and reduced number of Gr-1 with monocyte (i.e., non-polymorphonuclear) morphology ⁺ Cell correlation (r, s). EAE mean clinical score data represent three separate experiments for 10-20 mice/group. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Myocardial infarction data represent 2 experiments for at least 3 mice/group. Renal ischemia reperfusion data represent at least 4-5 mice/group of 2 experiments. DSS-colitis data represents at least 3 experiments in at least 3 mice/group. Image analysis was performed as described in materials and methods. Statistical analysis was performed using unpaired two-tailed student T test. When comparing PS-IMP groups with vehicle control groups, P was 0.05 (×0.01 (×0.001 (×0) P). Statistical analysis of the comparison was performed using a two-way analysis of variance (ANOVA) with post hoc Pad Prism 6.0 software. * P is p <0.05，**p<0.01，***p<0.001. For the sham control, n=5; day 1 each group n=7; for the control group, day 5 n=4-5 (since 3 animals died on days 3-4 we collected only one sample on about day 4.3, thus including the tubule scoring data) and day 5 negative microbead n=7.

Figure 6 shows that (a-P) IMP treatment significantly reduced inflammatory cytokine levels in WNV infected brains. Mice mimicking infection and WNV infection were treated with PS-IMP on day 6 post-immunization and brains were isolated and homogenized on day 7 post-immunization. Cytokine and chemokine levels were analyzed using a multiplex ELISA. Significantly reduced levels of CCL2 (a), IFN- γ (b), IL-6 (c), TNF (d), IL-10 (e), IL-4 (f), CCL3 (g), IL-12 (h), GM-CSF (k), IL-1α (l), IL-9 (n), IL-1β (o) and IL-2 (p) were observed in the brains of WNV infected animals treated with IMP as compared to the control. The levels of IL-3 (h), CCL5 (j) and IL-15 (m) were not significantly reduced upon IMP treatment.

Figure 7 shows that (a-F) WNV infected brains have little IMP present and that IMP is highly effective in reducing inflammatory monocytes in peritonitis. Brilliant blue NP or PS-IMP could not be detected on day 7 post immunization. WNV infected brain was analyzed immunohistochemically after injection on day 6 post immunization (a). Similarly, FITC-NP or PS-IMP (b) could not be detected by flow cytometry in WNV infected brains. PS-IMP was tested in the thioglycollic acid peritonitis model of C57BL/6 mice as shown in (C, d). Infusion of PS-IMP 24 hours after thioglycolate infusion reduced Ly6 isolation from the peritoneum ^Chi CD11 ^b+ (R3) number of monocytes (c, d). This effect was eliminated in splenic mice (e). Particle immunohistochemistry and flow cytometry data represent three separate experiments for at least 3 mice/group. Slides were counterstained with DAPI (blue) to identify nuclei. Thioglycolate data represents at least 3 experiments in 5 mice/group. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group.

Figure 8 shows that (a-G) IMP promotes accumulation of Φim in the spleen during epithelial repair in EAE, myocardial infarction and DSS-induced colitis. Significantly reduced CD45 was observed in EAE brain at day 14 post PLGA-IMP infusion ⁺ CD11b ⁺ Infiltration of monocyte-derived cells (a). The reduction of monocyte-derived cells in the brain compared to vehicle-treated controls corresponds to a significant accumulation of Φim in the spleen of PLGA-IMP treated animals (b). Significant accumulation of Φim in the spleen was observed in PLGA-IMP treated animals after myocardial infarction compared to vehicle treated controls (c, d). And H is ₂ Ki67 across the colon on day 9 of DSS challenge compared to the O control group ⁺ Immunohistochemical staining (f) revealed a characteristic Ki67 in the epithelium ⁺ Staining was relatively weak in DSS-challenged animals (f). Less crypt fallout was observed in DSS-challenged mice treated with PS-IMP (f). Furthermore, in these animals, ki67 ⁺ Dyeing is more common and intense in the lamina propria. Image analysis (g) of DSS-induced colitis vehicle compared to PS-IMP showed that the observed Ki67 differences were statistically significant. EAE data isMean ± standard deviation, and represent three separate experiments for 4-5 mice/group. Myocardial infarction data represent 2 experiments for at least 3 mice/group. DSS-colitis data represents at least 3 experiments in at least 3 mice/group. Image analysis was performed as described in materials and methods. Statistical analysis was performed using unpaired two-tailed student T test. When comparing PS-IMP groups with vehicle control groups, P was 0.05 (×0.01 (×0.001 (×0) P).

Figure 9 shows that conditioned IMP has a reduced ability to inhibit macrophage migration to the brain. Day 7 plasma was removed from mock or WNV infected animals and incubated with PS-IMP. On day 6 post immunization, these "conditioned" particles or unconditioned (untreated microbead) particles were reinfused into WNV infected mice. On day 7, inflammatory monocyte influx in the brain was examined by flow cytometry. Although untreated (normal IMP) resulted in reduced monocyte influx in the brain, the mock plasma coating IMP failed to inhibit monocyte migration. Of note, WNV serum coated IMP has a reduced ability to inhibit monocytes relative to uncoated IMP, but still has some reduced migration.

Fig. 10 shows that PLGA-IMP induces apoptosis of both inflammatory monocytes and neutrophils in TG model. Infusion of PLGA-IMP into thioglycolate-induced wild-type mice resulted in Ly6C expressing the apoptosis markers annexin V and caspase-3 ^hi Phi IM and Ly6G ⁺ The number of neutrophils increases significantly. This was not observed in the mock treated animals. Flow cytometry data are mean ± standard deviation and represent three separate experiments of 4-5 mice/group. Statistical analysis was performed using one-way anova and Tukey-Kramer post hoc test. When comparing the wild-type PS-IMP group with all other groups, p.ltoreq.0.05 (.

FIG. 11 shows that (A-B) apoptosis can be detected within 2 hours after PLGA or PS IMP infusion. Two hours after injection, PS-IMP was localized to cd11b+ly6c+ly6g-monocytes in the spleens of mock and TG peritonitis-induced mice. The uptake of PS-IMP is associated with the activity of the enzyme caspase-3, which is indicative of apoptosis (a). In PS-IMP treated, TG peritonitis induced mice, an increased number of monocytes were positive for caspase-3 activity (b).

Figure 12 shows that PLGA-IMP avoided inflammatory lesions in the myocardial infarction model. PLGA-IMP infusion for four days resulted in reduced infarct size, e.g., H, compared to vehicle-treated controls &E histological and image analysis. In addition, the reduced occlusion size in PLGA-IMP treated mice compared to vehicle treated controls was compared to fewer cd68+ macrophages and Ly6C in the spleen ^hi The increase in cells is correlated.

Figure 13 shows that PLGA-IMP infusion avoided inflammatory lesions in myocardial ischemia (LAD occlusion) -reperfusion model. The coronary arteries were occluded and blood flow was returned after 30 minutes. Mice were treated with PLGA-IMP for 4 days. Treatment with PLGA-IMP significantly reduced infarct scar size and systolic ejection fraction compared to PBS control animals.

Fig. 14 shows a micrograph of (a) poly (lactide-co-glycolide) (PLG) particles. B and C show characterization of the surface functionalized poly (lactide-co-glycolide) particles by dynamic light scattering analysis, including average size (nm) and zeta potential (mV) of PLG-PEMA particles. At 2.5X10 in 18.2M omega water on a Markov laser particle sizer (Malvern Zetasizer Nano ZS) (Markov instruments Inc. (Malvern Instruments, westborough, mass.)) ⁵ Count rate per second surface functionalized poly (lactide-co-glycolide) particles were analyzed. The population of surface functionalized poly (lactide-co-glycolide) particles varies from 5 to 15% per batch, but generally has a Z-average diameter of 567nm, a peak diameter of 670nm, and a polydispersity index of 0.209.

Fig. 15 shows (a) shows that PLGA-IMP infusion avoided inflammatory lesions in EAE. After cessation of treatment, there is a disease free period, however, over time, the disease recurs. (B) PLGA-IMP infusion (rather than vehicle control) resulted in expansion and induction of regulatory T cells expressing anti-inflammatory proteins such as PD-1. (C) These tregs are most abundant during IMP treatment and decline over time after treatment ceases.

Fig. 16 shows (a) removal of pathogenic proteins and cell debris from the blood of a subject with an inflammatory disorder, and (B) concentration and presentation of regulatory proteins from the blood stream of a subject with an inflammatory disorder.

Fig. 17 shows that exemplary negatively charged particles are capable of determining the presence or absence of proteins from the serum of a subject to determine whether the subject is experiencing an inflammatory process.

Detailed Description

The inventors surprisingly found that when negatively charged particles, such as polystyrene, PLGA or diamond particles of a certain size and zeta potential, are administered to a subject, the inflammatory immune response is improved. In addition, the inventors have surprisingly found that these same negatively charged particles, when administered to a subject, induce apoptosis and improved clearance of monocytes and/or neutrophils in the subject. It is also surprising that these same negatively charged particles, when administered to a subject, induce tolerogenic dendritic cells, suppressor cells, and regulatory T cells. It has also been found that these negatively charged particles are capable of removing pro-inflammatory mediators from the inflammatory environment. Similarly, negatively charged particles may also concentrate and present regulatory mediators to further induce regulatory T cell responses or otherwise ameliorate inflammatory immune responses. Thus, negatively charged particles are useful in the treatment of any disease or disorder characterized by excessive inflammatory immune response, such as autoimmune diseases, as well as in the treatment of bacterial and viral infections. Finally, particles embedded with one or more antigens may induce antigen-specific tolerance in the subject.

As used herein, "particle" refers to any composition of matter of non-tissue origin that is a microsphere or microspheroidal entity or bead. The term "particle" and the term "microbead" are used interchangeably. In addition, the term "particle" may be used to encompass microbeads and microspheres.

"carboxylated particles" or "carboxylated microbeads" or "carboxylated microspheres" include any particle modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups enhances uptake of the particles by phagocytes/monocytes from the blood circulation, for example, by interaction with scavenger receptors such as MARCO. Carboxylation may be achieved using any compound capable of adding a carboxyl group, including but not limited to poly (ethylene-maleic anhydride) (PEMA).

"negatively charged particles" or "negatively charged microbeads" or "negatively charged microspheres" include any particle that has a negative charge in itself or is modified to have a negative charge. In some embodiments, the particles have a negative charge by carboxylation of the particles.

An "antigenic moiety" as used herein refers to any moiety, such as a peptide, that is recognized by the immune system of the host. Examples of antigenic moieties include, but are not limited to, autoantigens and/or bacterial or viral proteins, peptides or components. Without being bound by theory, although negatively charged microbeads themselves may be recognized by the immune system, negatively charged microbeads having no other substances attached thereto are not considered "antigenic moieties" for the purposes of the present invention.

As used herein, "naked microbeads" or "naked particles" or "naked microspheres" refer to microbeads, particles, or microspheres that are not carboxylated or otherwise modified.

As used herein, "pro-inflammatory mediator" or "pro-inflammatory polypeptide" refers to a polypeptide or fragment thereof that induces, maintains, or delays inflammation in a subject. Examples of pro-inflammatory mediators include, but are not limited to, cytokines and chemokines.

As used herein, an "inflammatory environment" or "inflammatory lesion" refers to a site in a subject where inflammation or an increase in the concentration of a pro-inflammatory mediator occurs. The inflammatory environment may encompass the entire subject's blood circulation. For example, when a subject suffers from a systemic inflammatory disorder, inflammatory mediators can be found throughout the subject's blood circulation. Thus, in these embodiments, the inflammatory environment is not contained within the discrete region.

By "regulatory T cells" or "tregs" or "suppressor T cells" is meant T cells capable of modulating a T cell immune response. For example, tregs may be capable of inhibiting CD4 ⁺ Or CD8 ⁺ CD4 of effector function of T cell response ⁺ Or CD8 ⁺ T cells.

The particles may have any particle shape or conformation. However, in some embodiments, it is preferable to use particles that are less likely to agglomerate in vivo. Examples of particles within these embodiments are those having a spherical shape.

It is not necessary that each particle be uniform in size, but the particles must generally be of a size sufficient to trigger phagocytosis in antigen presenting cells or other MPS cells. Preferably, the particle size is on the order of micro-or nano-scale to enhance solubility, avoid complications that may result from aggregation in vivo, and facilitate pinocytosis. Particle size may be a factor in the area from interstitial space uptake to lymphocyte maturation. Particles having an average diameter of about 0.1 μm to about 10 μm are capable of triggering phagocytosis. Thus, in one embodiment, the particles have diameters within these limits. In another embodiment, the particles have an average diameter of about 0.2 μm to about 2 μm. In another embodiment, the particles have an average diameter of about 0.3 μm to about 5 μm. In yet another embodiment, the particles have an average diameter of about 0.5 μm to about 3 μm. In another embodiment, the particles have an average size of about 0.1 μm, or about 0.2 μm, or about 0.3 μm, or about 0.4 μm, or about 0.5 μm, or about 1.0 μm, or about 1.5 μm, or about 2.5 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm. In a specific embodiment, the particles have a size of about 0.5 μm. The particles in the composition need not have a uniform diameter. For example, the pharmaceutical formulation may comprise a plurality of particles, some of which are about 0.5 μm and others of which are about 1.0 μm. Any mix of particle sizes within these given ranges will be useful.

In some embodiments, the particles are nonmetallic. In these embodiments, the particles may be formed from a polymer. In a preferred embodiment, the particles are biodegradable in the individual. In this embodiment, the particles may be provided in multiple doses in the individual without the particles accumulating in the individual. Examples of suitable particles include polystyrene particles, PLGA particles, PLURIONICS stabilized polypropylene sulfide particles, and diamond particles.

Preferably, the particle surface is composed of a material that minimizes non-specific or unwanted biological interactions. Particle surfaceInteractions with the interstitium may be a factor in lymphatic absorption. The particle surface may be coated with a material that prevents or reduces non-specific interactions. By using hydrophilic layers such as poly (ethylene glycol) (PEG) and copolymers thereof such asCoating particles with a copolymer comprising poly (ethylene glycol) -bl-poly (propylene glycol) -bl-poly (ethylene glycol) results in steric stabilization, which reduces non-specific interactions with interstitial proteins, as demonstrated by improved lymphatic absorption following subcutaneous injection. All these facts indicate the importance of the physical properties of the particles in lymphatic absorption. Biodegradable polymers can be used to make all or some of the polymers and/or particles and/or layers. Biodegradable polymers can degrade, for example, by the reaction of functional groups with water in solution. The term "degrading" as used herein means becoming soluble by reducing the molecular weight or by converting hydrophobic groups into hydrophilic groups. Polymers having ester groups typically undergo spontaneous hydrolysis, for example, polylactides and polyglycolides.

The particles of the present invention may also contain additional components. For example, the carrier may have an imaging agent incorporated into or conjugated to the carrier. An example of a currently commercially available carrier nanosphere with imaging agent is a Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals, known as Quantum Dots (QDs), have become ideal donors in FRET applications: their high quantum yield and tunable size-dependent stokes shift allow different sizes to emit blue to infrared light when excited at a single ultraviolet wavelength. (Bruchez, et al, science,1998,281,2013 (Bruchez et al, science,1998, volume 281, page 2013)), niemeeyer, C.M Angew.chem.Int.Ed.2003,42,5796 (Niemeeyer, C.M, international edition of applied chemistry, 2003, volume 42, page 5796), wagger, A.methods enzymol.1995,246,362 (Wagger, A., "methods of enzymology, 1995, volume 246, page 362)), blus, L.E.J.chem.Physics 1993,79,5566 (J.S, L.E., 1993, volume 79, page 5566). Quantum dots, such as hybrid organic/inorganic quantum dots based on a class of polymers known as dendrimers, are useful in biomarker, imaging, and optical biosensing systems. (Lemon, et al, J.am.chem.Soc.2000,122,12886 (Lemon et al, society of chemical Co., ltd., 2000, volume 122, page 12886)). Unlike the synthesis of traditional inorganic quantum dots, the synthesis of these mixed quantum dot nanoparticles does not require high temperature or highly toxic, unstable reagents. (Etiene, et al, appl. Phys. Lett.87,181913,2005 (Etiene et al, applied physics report, volume 87, page 181913, 2005)).

The particles may be formed from a wide variety of materials. The particles are preferably composed of a material suitable for biological use. For example, the particles may be composed of glass, silica, polyester of hydroxycarboxylic acid, polyanhydride of dicarboxylic acid, or copolymer of hydroxycarboxylic acid and dicarboxylic acid. More generally, the carrier particles may be composed of: linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or polyester of alkoxyhydroxy acids, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or polyanhydride of alkoxydicarboxylic acids. In addition, the carrier particles may be, or consist of, quantum dots, such as quantum dot polystyrene particles (Joumaa et al (2006) Langmuir 22:1810-6 (Joumaa et al, 2006, langmuir, volume 22, pages 1810-6)). Carrier particles comprising a mixture of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) may also be employed. For example, the carrier particles may comprise materials including: polyglycolic acid Polymer (PGA), polylactic acid Polymer (PLA), polysebacic acid Polymer (PSA), poly (lactic-co-glycolic acid) copolymer (PLGA), poly (lactic-co-sebacic acid) copolymer (PLSA), poly (glycolic acid-co-sebacic acid) copolymer (PGSA), polypropylene polysulfide polymer, poly (caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates And polymers or copolymers of degradable polyurethanes, and copolymers thereof with linear or branched, substituted or unsubstituted alkyl, haloalkyl, sulfanyl, aminoalkyl, alkenyl, or aromatic hydroxy acids or dicarboxylic acids. In addition, biologically important amino acids having reactive side chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or enantiomers thereof, may be included in copolymers with any of the above materials to provide reactive groups for conjugation with antigenic peptides and proteins or conjugate moieties. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene polysulfide polymer and PLGA polymer. Biocompatible but non-biodegradable materials may also be used in the carrier particles of the present invention. For example, acrylates, ethylene-vinyl acetate, acyl substituted cellulose acetate, nondegradable polyurethanes, styrene, vinyl chloride, vinyl fluoride, vinylimidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol,(DuPont, wilmington, del.) and nylon.

In one embodiment, the buffer solution in contact with the immunomodifying particle may have an alkaline pH. Suitable alkaline pH of the alkaline solution includes 7.1, 7.5, 8.0, 8.5, 9.5, 10.0.5, 11.0, 11.5, 12.0, 12.5, 13.0 and 13.5. The buffer solution may also be made of any suitable base and conjugates thereof. In some embodiments of the present invention, the buffer solution may include, but is not limited to, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate, and conjugates thereof.

In one embodiment of the invention, the immunomodifying particle comprises a copolymer. These copolymers may have different molar ratios. Suitable copolymer ratios for the immunomodified particles of the present invention may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91; 9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In another embodiment, the copolymer may be a periodic, statistical, linear, branched (including star, brush, or comb copolymers) copolymer. In some embodiments, the copolymer ratio may be, but is not limited to, polystyrene: poly (vinyl carboxylate)/80:20, polystyrene: poly (vinyl carboxylate)/90:10, poly (vinyl carboxylate): polystyrene/80:20, poly (vinyl carboxylate): polystyrene/90:10, polylactic acid: polyglycolic acid/50:50, polylactic acid: polyglycolic acid/80:20, or polylactic acid: polyglycolic acid/90:10.

The particles of the present invention may be manufactured by any means generally known in the art. Exemplary methods of making particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, precipitation polymerization, emulsion volatilization, emulsion diffusion, solvent displacement, and salting out (Astete and Sabliov, J.Biomate.Sci.Polymer Edn.,17:247-289 (2006) (Astete and Sabliov, J.Biol.Material science-Polymer edition, 17, pages 247-289, 2006)). Manipulation of the manufacturing process of PLGA particles can control particle properties (e.g., size distribution, zeta potential, morphology, hydrophobicity/hydrophilicity, polypeptide entrapment, etc.). The size of the particles is affected by a variety of factors including, but not limited to, the concentration of PLGA, the solvent used to make the particles, the nature of the organic phase, the surfactant used in the manufacture, the viscosity of the continuous and discontinuous phases, the nature of the solvent used, the temperature of the water used, sonication, evaporation rate, additives, shear stress, sterilization, and any antigen or polypeptide encapsulating properties.

Particle size is affected by polymer concentration; larger particles are formed from higher polymer concentrations. For example, when using propylene carbonate solvent, an increase in PLGA concentration from 1% to 4% (w/v) can increase the average particle size from about 205nm to about 290nm. Alternatively, in ethyl acetate and 5% Pluronic F-127, an increase in PLGA concentration from 1% to 5% (w/v) increases the average particle size from 120nm to 230nm.

The viscosity of the continuous and discontinuous phases is also an important parameter that affects the diffusion process, a critical step in forming smaller particles. The size of the particles increases with increasing viscosity of the dispersed phase, while the size of the particles decreases with the more viscous continuous phase. Generally, the lower the phase ratio of organic solvent to aqueous solvent, the smaller the particle size.

The homogenizer speed and agitation also affect particle size; generally, higher speeds and agitation will reduce the particle size, but there is a point where further increases in speed and agitation will not reduce the particle size. The size reduction is advantageously affected when the emulsion is homogenized with a high pressure homogenizer, compared to just high speed stirring. For example, at a 20% ratio in 5% PVA, the average particle size with stirring is 288nm, while the average particle size with homogenization (300 bar high pressure) is 231nm.

Important size reduction of the particles can be achieved by: the temperature of the added water was varied to improve the diffusion of the solvent. The average particle size decreases with increasing water temperature.

The nature of the polypeptide encapsulated in the particle also affects the particle size. Generally, encapsulation of a hydrophobic polypeptide results in the formation of smaller particles than encapsulation of a more hydrophilic polypeptide. The entrapment of more hydrophilic polypeptides is improved during the re-emulsification process by using high molecular weight PLGA and a high molecular weight first surfactant that causes a higher internal phase viscosity. Interactions between the solvent, polymer and polypeptide affect the efficiency of incorporation of the polypeptide into the particle.

The PLGA molecular mass affects the final average particle size. Generally, the higher the molecular mass, the larger the average particle size. For example, when the composition and molecular mass of PLGA varies (e.g., 12 to 48kDa for 50:50 PLGA; 12 to 98kDa for 75:25 PLGA), the average particle size also varies (about 102nm-154nm; about 132nm to 152nm, respectively). Even when the particles are of the same molecular mass, their composition can affect the average particle size; for example, particles having a 50:50 ratio generally form smaller particles than particles having a 75:25 ratio. The end groups on the polymer also affect particle size. For example, particles prepared with ester end groups formed particles having an average size of 740nm (pi=0.394), in contrast to 240nm (pi=0.225) for acidic PLGA end groups.

The solvent used may also affect the particle size; solvents that reduce the surface tension of the solution also reduce particle size.

The organic solvent is removed by vacuum evaporation to avoid polymer and polypeptide damage and to promote final particle size reduction. Vacuum evaporation of the organic solvent can be more efficient in forming smaller particles. For example, vacuum evaporation produces an average particle size that is about 30% smaller than the average particle size produced at normal evaporation rates.

The amplitude of the sonication wavelength also affects the particle characteristics. The amplitude of the wavelength should be over 20% and sonicated for 600 to 800s to form a stable miniemulsion without any change in droplet size. However, the main disadvantage of sonication is that the emulsion formed does not have monodispersity.

Organic phases that can be used to produce the particles of the present invention include, but are not limited to, ethyl acetate, methyl ethyl ketone, propylene carbonate, and benzyl alcohol. The continuous phase that can be used includes, but is not limited to, the surfactant poloxamer 188.

A variety of surfactants can be used in making the particles of the present invention. The surfactant may be anionic, cationic or nonionic. Surfactants in the poloxamer and poloxamer families are commonly used for particle synthesis. Surfactants that may be used include, but are not limited to, PEG, tween-80, gelatin, dextran, pluronic L-63, PVA, methylcellulose, lecithin, and DMAB. In addition, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (D-alpha-tocopheryl polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are required (e.g., in a multiple emulsion volatilization process). The two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

Solvents useful in producing the particles of the present invention include, but are not limited to, acetone, tetrahydrofuran (THF), chloroform, and members of the chloride family, methyl chloride. The choice of organic solvent requires two selection criteria: the polymer must be soluble in the solvent and the solvent must be fully miscible with the aqueous phase.

Salts useful in producing the particles of the present invention include, but are not limited to, magnesium chloride hexahydrate, magnesium acetate tetrahydrate.

Common salting-out agents include, but are not limited to, electrolytes (e.g., sodium chloride, magnesium acetate, magnesium chloride) or non-electrolytes (e.g., sucrose).

The stability and size of the particles of the present invention can be improved by the addition of compounds including, but not limited to, fatty acids or short carbon chains. The addition of lauric acid with a longer carbon chain is associated with an improvement in the particle characteristics. Furthermore, the addition of hydrophobic additives may improve particle size, incorporation of the polypeptide into the particle, and release characteristics. The formulation of the particles may be stabilized by lyophilization. The addition of cryoprotectants such as trehalose may reduce aggregation of the particles upon lyophilization.

Suitable microbeads that are currently commercially available include polystyrene microbeads, such as FluoSpheres (Molecular Probes, eugene, oreg.).

Physical properties also relate to the applicability of the nanoparticle after uptake and retention in areas with immature lymphocytes. They include mechanical properties such as rigidity or elasticity. Some embodiments are based on an elastic core, such as a poly (propylene sulfide) (PPS) core with a coating, such as a hydrophilic coating, as in PEG, such as in PPS-PEG systems recently developed and characterized for systemic (but not targeted or immune) delivery. The elastic core is in contrast to a substantially rigid core, as in polystyrene or metal nanoparticle systems. The term elastic refers to some elastic material other than natural or synthetic rubber, while elastic is a term familiar to those skilled in the polymer arts. For example, cross-linked PPS may be used to form the hydrophobic elastic core. PPS is a polymer that degrades under oxidative conditions to a sulfoxide and eventually forms a polysulfone, transitioning from a hydrophobic rubber to a hydrophilic water-soluble polymer. Other sulfide polymers may be suitable for use, wherein the term sulfide polymer refers to a polymer having sulfur in the backbone of the matrix. Other elastomeric polymers that may be used are polyesters having glass transition temperatures below about 37 ℃ under hydration conditions. The hydrophobic core may be advantageously used with a hydrophilic cover layer, as the core and cover layer may tend not to mix and thus the cover layer tends to spatially expand away from the core. Core refers to particles having a layer thereon. Layer refers to a material that covers at least a portion of the core. The layers may be adsorbed or covalently bonded. The particles or cores may be solid or hollow. Elastic hydrophobic cores are preferred over rigid hydrophobic cores, such as crystalline or glass (as in the case of polystyrene) cores, because particles with elastic hydrophobic cores can carry higher hydrophobic drug loadings.

Another physical property is the hydrophilicity of the surface. The hydrophilic material may have a solubility in water of at least 1 gram per liter when it is uncrosslinked. Steric stabilization of particles with hydrophilic polymers can enhance uptake from the matrix by reducing non-specific interactions; however, the increased stealth properties of the particles may also reduce internalization of phagocytes in areas with immature lymphocytes. However, challenges have been encountered to balance these conflicting features, and the present application describes the generation of nanoparticles for efficient lymphatic delivery to DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic component, such as a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of the following: polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids and polyethers. The molecular weight of the polymer in the layer may be adjusted to provide a useful degree of steric hindrance in vivo, for example from about 1,000 to about 100,000 or even more; the skilled person will immediately understand that all ranges and values within the explicitly stated ranges are conceivable, for example between 10,000 and 50,000.

The composition of the particles has been found to affect the length of time the particles remain in the body and are resistant to the requirements of rapid particle uptake and clearance/degradation. Due to over 50:50 lactide: the glycolide ratio slows the degradation rate and the particles of the application have lactide of about 50:50 or less: glycolide ratio. In one embodiment, the particles of the present application have about 50:50d, l-lactide: glycolide ratio.

The particles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles; they facilitate reaction with other molecules. Examples of nucleophiles are primary amines, thiols and hydroxyl groups. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

The efficacy of colloidal therapeutic agents such as the negatively charged particles of the present invention is closely related to the in vivo distribution of the particles. The distribution of the colloidal system can be predicted by measuring the zeta potential. Zeta potential is a measure of the potential difference between the dispersing medium and the layer of stabilizing fluid attached to the dispersed particles and indicates the degree of repulsion between adjacent, similarly charged particles in the dispersion. A high zeta potential is indicative of the stability and good dispersibility of the colloidal formulation. In a preferred embodiment, the zeta potential of the pharmaceutical formulation of the invention is indicative of good dispersibility of the formulation in vivo.

The particles of the present invention may have a specific zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than about-100 mV. In one embodiment, the zeta potential is less than about-50 mV. In certain embodiments, the particles have a zeta potential of between-100 mV and 0mV. In another embodiment, the particles have a zeta potential of between-75 mV and 0mV. In another embodiment, the particles have a zeta potential of between-60 mV and 0mV. In another embodiment, the particles have a zeta potential of between-50 mV and 0mV. In yet another embodiment, the particles have a zeta potential of between-40 mV and 0mV. In another embodiment, the particles have a zeta potential of between-30 mV and 0mV. In another embodiment, the particles have a zeta potential of between-20 mV and +0 mV. In another embodiment, the particles have a zeta potential of between-10 mV and-0 mV. In another embodiment, the particles have a zeta potential of between-100 mV and-50 mV. In another embodiment, the particles have a zeta potential of between-75 mV and-50 mV. In another embodiment, the particles have a zeta potential of between-50 mV and-40 mV.

The particles of the invention may be administered in any dose effective to inhibit an inflammatory immune response in a subject in need thereof, or to treat a bacterial or viral infection in a subject in need thereof. In certain embodiments, about 10 is provided to an individual ² To about 10 ²⁰ And (3) particles. In another embodiment, about 10 is provided ³ To about 10 ¹⁵ Particles between each. In yet another embodimentIn providing about 10 ⁶ To about 10 ¹² Particles between each. In yet another embodiment, about 10 is provided ⁸ To about 10 ¹⁰ Particles between each. In a preferred embodiment, the preferred dosage is 0.1% solids/ml. Thus, for 0.5 mu microbeads, the preferred dose is about 4.gtoreq.10 ⁹ The preferred dosage for 0.05 mu microbeads is about 4.gtoreq.10 per microbead ¹² For each bead, for 3 mu beads, the preferred dosage is 2.gtoreq.10 ⁷ And (3) microbeads. However, the present invention encompasses any dose effective to treat the particular condition to be treated.

The invention is useful for treating immune related disorders such as autoimmune diseases, transplant rejection, inflammatory diseases and/or disorders, ischemia reperfusion, stroke, myocardial infarction and allergies. Substitution of synthetic biocompatible particle systems to induce immune tolerance can lead to easier manufacture, expanded availability of therapeutic agents, increased uniformity between samples, increased number of potential treatment sites, and significantly reduced likelihood of allergic reactions to carrier cells.

As used herein, the term "immune response" includes T cell-mediated and/or B cell-mediated immune responses. Exemplary immune responses include T cell responses such as cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine responsive cells such as macrophages. Immune cells involved in immune response include lymphocytes, such as B cells and T cells (CD 4 ⁺ 、CD8 ⁺ Th1 and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; bone marrow cells such as macrophages, eosinophils, mast cells, basophils and granulocytes. In some embodiments, the modified particles of the invention are effective to reduce inflammatory cells transported to the site of inflammation.

As used herein, the term "inflammatory monocyte" refers to any bone marrow cell that expresses any combination of CD14/CD16 and CCR 2. As used herein, the term "inhibitory neutrophils" encompasses monocyte-derived suppressor cells and/or neutrophils.

As used herein, the term "non-responsive," "tolerogenic," or "antigen-specific tolerogenic" refers to T cells that are insensitive to T cell receptor-mediated stimuli. This insensitivity is typically antigen specific and persists after cessation of exposure to the antigen peptide. For example, T cell unresponsiveness is characterized by a lack of cytokine production, such as IL-2. T cell unresponsiveness can occur when T cells are exposed to antigen and receive a first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of a co-stimulatory molecule) results in the inability to produce cytokines, and subsequent inability to proliferate. Therefore, no cytokine production can prevent proliferation. However, non-responsive T cells can proliferate if cultured with cytokines (e.g., IL-2). For example, a lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines, can also be observed. Alternatively, reporter constructs may be used. For example, non-responsive T cells are unable to initiate DL-2 gene transcription induced by heterologous promoters under the control of the 5' IL-2 gene enhancer or by API sequence multimers that can be found in enhancers (Kang et al 1992science 257:1134 (Kang et al 1992, science 257, p 1134)).

As used herein, the term "tolerizing" refers to a method performed on a portion of a subject receiving treatment as compared to an untreated subject, wherein: a) A reduced level of a specific immune response (thought to be mediated at least in part by antigen specific effector T lymphocytes, B lymphocytes, antibodies, or equivalents thereof); b) Delay in onset and progression of specific immune responses; or c) a reduced risk of onset or progression of a specific immune response. "specific" immune tolerance occurs when immune tolerance against certain antigens is preferentially elicited over others. "nonspecific" immune tolerance occurs when there is no difference in the initiation of immune tolerance against the antigen that causes the inflammatory immune response. "quasi-specific" immune tolerance occurs when immune tolerance is elicited semi-differentially against antigens that lead to pathogenic immune responses but not against other antigens that lead to protective immune responses.

One representative of tolerogenic activity is the ability of the particles to stimulate the production of appropriate cytokines at the target site. Immunomodulatory cytokines released by T-suppressor cells at target sites are known as TGF-beta (Miller et al, proc. Natl. Acad. Sci. USA 89:421,1992 (Miller et al, proc. Natl. Acad. Sci. USA, vol. USA 89, vol. 421, 1992)). Other factors that may be produced during tolerance are cytokines IL4 and IL-10, and mediator PGE. In contrast, lymphocytes in tissues undergoing active immune destruction secrete cytokines such as IL-I, IL-2, IL-6 and IFNγ. Thus, the efficacy of the modified particles can be evaluated by measuring their ability to stimulate the appropriate type of cytokine.

In view of this, an animal model system can be used to conduct rapid screening tests for modifying the effective pattern and schedule of particles, effective mucoadhesive components, effective combinations, or mucosal administration. The mucosal surfaces of animals are treated with the test particle composition and sometimes challenged by administration of a pathogenic antigen or infectious pathogen. Spleen cells were isolated and cultured in vitro in the presence of a pathogenic antigen or an antigen derived from an infectious pathogen at a concentration of about 50 μg/mL. Cytokine secretion into the culture medium can be quantified by standard immunoassays.

The ability of particles to inhibit cellular activity can be determined using cells isolated from animals immunized with modified particles, or by establishing cell lines that respond to pathogenic or viral antigen target antigens (Ben-Nun et al, eur. J. Immunol.11:195,1981 (Ben-Nun et al, european journal of immunology, volume 11, page 195, 1981)). In one variation of this experiment, light irradiation (about 1000 to 1250 rad) inhibited the population of cells to prevent proliferation, the inhibited cells were cultured with the responder cells, and then the proliferative activity of the responder cells was quantified using tritiated thymidine incorporation (or MTT). In another variation, the suppressor cell and responder cell populations are cultured in upper and lower layers of a dual-chamber transwell culture system (Costar, cambridge mass) that allows the cell populations to co-incubate within 1mm of each other separated by a polycarbonate membrane (WO 93/16724). In this method, irradiation to inhibit the cell population is unnecessary, as proliferation activity of the responsive cells can be measured separately.

The effectiveness of the compositions and modes of administration for treating a particular disease can also be demonstrated in a corresponding animal disease model. The ability of the treatment to attenuate or delay symptoms of the disease is monitored at the level of circulating biochemical and immunological markers of the disease, immunohistology of the affected tissue, and overall clinical characteristics appropriate for the model employed. Non-limiting examples of animal models that can be used for testing are included in the following sections.

The present invention contemplates modulating tolerance by modulating the TH1 response, the TH2 response, the TH17 response, or a combination of these responses. Modulation of TH1 response encompasses altering, for example, the expression of interferon-gamma. Modulating the TH2 response encompasses altering expression of, for example, IL-4, IL-5, IL-10, and any combination of IL-13. Typically an increase (decrease) in TH2 response will include an increase (decrease) in expression of at least one of IL-4, IL-5, IL-10, or IL-13; more typically an increase (decrease) in TH2 response will comprise an increase in the expression of at least two of IL-4, IL-5, IL-10, or IL-13, most typically an increase (decrease) in TH2 response will comprise an increase in at least three of IL-4, IL-5, IL-10, or IL-13, and ideally an increase (decrease) in TH2 response will comprise an increase (decrease) in the expression of all of IL-4, IL-5, IL-10, and IL-13. Modulation of TH17 encompasses altering, for example, the expression of TGF- β, IL-6, IL-21, and IL-23, and the effector levels of IL-17, IL-21, and IL-22.

Tolerance to autoantigens and autoimmune diseases is through a variety of mechanisms including negative selection of autoreactive T cells in the thymus, as well as peripheral tolerance of autoreactive T cells that escape thymus clearance and are found in the peripheryA mechanism. Examples of mechanisms that provide peripheral T cell tolerance include "ignoring" of self-antigens, non-response or non-response to self-antigens, cytokine immune bias, and activation-induced cell death of autoreactive T cells. In addition, regulatory T cells have been shown to be involved in mediating peripheral tolerance. See, e.g., walker et al (2002) Nat.Rev.Immunol.2:11-19 (Walker et al, 2002, nature review: immunology, volume 2, pages 11-19); shevach et al (2001) Immunol. Rev.182:58-67 (Shevach et al, 2001, J. Immunol. 182, pages 58-67). In some cases, peripheral tolerance to self-antigens is lost (or destroyed) and then autoimmune reactions occur. For example, in animal models of EAE, activation of Antigen Presenting Cells (APCs) by TLR innate immune receptors has been shown to disrupt self-tolerance and lead to induction of EAE (Waldner et al (2004) J.Clin. Invest.113:990-997 (Waldner et al, 2004, J.clinical study, volume 113, pages 990-997)). In certain embodiments, the particles of the invention are capable of inducing tolerance or systemic immunomodulation by increasing the frequency and/or effector function of tregs. The Treg substantially upregulated by the particles of the invention is CD4 ⁺ CD25 ⁺ Treg, which also expresses the transcription repressor fork P3 (FoxP 3). However, tregs upregulated by the particles of the invention may also be CD8 ⁺ Treg or suppressor cells.

Thus, in some embodiments, the invention provides methods for increasing antigen presentation while inhibiting or reducing TLR7/8, TLR9, and/or TLR 7/8/9-dependent cell stimulation. As described herein, administration of the specifically modified particles results in antigen presentation by the DC or APC while inhibiting TLR7/8, TLR9, and/or TLR7/8/9 dependent cellular responses associated with the immunostimulatory polynucleotide. Such inhibition may include a decrease in the level of one or more TLR-related cytokines.

In another aspect of the invention, the negatively charged particles act as a molecular sink to remove pro-inflammatory mediators, pathological proteins, and cell debris from the blood of a subject with an inflammatory response, as depicted in fig. 16A. Alternatively or additionally, negatively charged particles of the invention may pass through the junctionThe regulatory proteins in the blood of subjects with inflammatory responses are synthesized to concentrate the regulatory proteins and present these to their cognate receptors to further improve the immune response, as depicted in fig. 16B. As discussed in more detail below, the inventors have found that at least 15 serum proteins are found to bind to negatively charged particles in subjects with an active inflammatory immune response, while no negatively charged particles are found to bind to untreated subjects subject) or a subject who is not undergoing an immune response. This can be used not only in the above methods, where the particles can act as a pro-inflammatory depot or as a concentrator/presenter of the immunomodulator, but also in the case of diagnostic methods. In particular, the negatively charged particles of the present invention can be used in large scale diagnostic methods for blood samples in cases where other methods such as mass spectrometry and other proteomic methods have failed. Surprisingly it was found that when inflammatory plasma or serum is incubated with negatively charged particles as described herein, this results in binding and subsequent purification of proteins in serum/plasma that are not present in non-inflammatory or homeostatic conditions.

In another aspect of the invention, particles comprising an antigen are provided. Nanoparticles carrying antigens on their surface have been successfully used to induce T cell tolerance (Getts et al, 2012Nature Biotechnology 30:1217-1223 (Getts et al, 2012, nature Biotechnology, volume 30, pages 1217-1223)). Tolerance induced by peptide-coupled particles depends on both the induction of T cell unresponsiveness and the activity of regulatory T cells, and may represent an alternative way of treating autoimmune disorders by inducing T cell tolerance. This T cell tolerance was observed when peptide coupling to biodegradable (PLG) particles was used.

In one embodiment, the particles of the invention are coupled to an antigen comprising one or more epitopes associated with an allergic reaction, an autoimmune disease, and/or an inflammatory disease or disorder. An antigen may comprise one or more copies of an epitope. In one embodiment, the antigen comprises a single epitope associated with a disease or disorder. In another embodiment, the antigen comprises more than one epitope associated with the same disease or disorder. In yet another embodiment, the antigen comprises more than one epitope associated with a different disease or disorder. In another embodiment, the antigen comprises one or more epitopes associated with one or more allergies. In another embodiment, the antigen comprises one or more epitopes associated with multiple sclerosis, type 1 diabetes, celiac disease, and/or inflammatory bowel disease (including crohn's disease or ulcerative colitis).

In one embodiment, the epitope is from myelin basic protein (e.g., SEQ ID NO:4975 and 4976), proteolipid protein (e.g., SEQ ID NO: 4977), myelin oligodendrocyte glycoprotein (e.g., SEQ ID NO:1 and 4978), aquaporin (e.g., SEQ ID NO: 4979), myelin-associated glycoprotein (e.g., SEQ ID NO: 4980), insulin (e.g., SEQ ID NO: 4981), glutamate decarboxylase (e.g., SEQ ID NO: 4982), gliadin (e.g., SEQ ID NO:4983-4985, or 5136-5140), or the alpha 3 chain of type IV collagen (e.g., SEQ ID NO: 5017), or fragments, homologs, or subtypes thereof. In another embodiment, the epitope is from gluten, including from gliadin and/or glutenin. In one embodiment, the epitope is from an insulin homolog, such as those described in U.S. patent No.8,476,228, which is hereby incorporated in its entirety for all purposes. In one embodiment, gliadin epitopes are those described in SEQ ID NO 13, 14, 16, 320 or 321 in U.S. patent application No.20110293644, or Sollid et al (2012) Immunogenetics 65:455-460 (Sollid et al 2012, immunogenetics, volume 65, pages 455-460), both of which are hereby incorporated by reference in their entirety for all purposes.

Further non-limiting examples of epitopes associated with various autoimmune and/or inflammatory diseases or disorders contemplated by the present invention are described in tables 1 and 2.

TABLE 1 representative Linear epitopes

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Not all epitopes are linear epitopes; epitopes may also be discontinuous conformational epitopes. A variety of discrete epitopes associated with autoimmune or inflammatory diseases and/or disorders are known. Non-limiting examples of discontinuous epitopes are described in table 2.

TABLE 2 representative discontinuous epitopes

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In another aspect of the invention, particles encapsulating an antigen are provided. Particles that encapsulate an antigen within a particle can be used to induce T cell tolerance in a subject. Examples of antigens that may be encapsulated within the particles of the invention include, but are not limited to, exogenous antigens such as viral and bacterial antigens, endogenous antigens, autoantigens, tumor antigens, and/or natural antigens.

Monocytes and macrophages play a central role in the initiation and regression of inflammation, mainly by phagocytosis, inflammatory cytokines, release of reactive oxygen species and activation of the acquired immune system (Auffray et al 2009Annu Rev Immunol 27:669-692 (a)ufray et al, 2009, "immunology annual book", volume 27, pages 669-692)). Typically, monocytes circulate in the blood stream for a short period of time after which apoptosis occurs, however, the stimulus signal may trigger monocyte survival by inhibiting the apoptotic pathway, thereby helping to maintain the inflammatory response. The mode of action of the anti-apoptotic proteins is to inhibit activation of caspases or apoptotic programs. Phosphatidylinositol-3 kinase (PI-3K)/Akt, ERK, fas, TNF, heat shock proteins, and anti-apoptotic molecules, among others, play a key role in determining monocyte longevity. During the inflammatory response, inflammatory cells such as monocytes and macrophages are recruited to the site of inflammation. This recruitment is critical for effective control and clearance of infection, but the recruited monocytes also contribute to the onset of inflammatory and degenerative diseases. The accumulation of monocytes can be detrimental and exacerbate diseases such as atherosclerosis, arthritis and multiple sclerosis. Resolution of inflammation requires reduction and/or inhibition of inflammatory cell entry into inflammatory foci, and apoptosis of inflammatory cells already exists. Apoptotic caspases play a fundamental role by proteolytic cleavage of cells by degradation of proteins with a variety of biological functions. For example, caspase-3 activation is for CD14 ⁺ Apoptosis of monocytes is critical (Fahy et al, 1999J. Immunol.163:1755-1762 (Fahy et al, 1999, J. Immunol.163, pages 1755-1762)).

Negatively charged particles (sometimes referred to herein as "immune-modified particles" or "IMPs") of the present invention specifically inhibit migration of inflammatory monocytes into inflammatory lesions. Inflammatory monocytes ingest IMP in a collagen-like structure macrophage receptor (MARCO) dependent manner and migrate to the spleen where they undergo caspase 3 mediated cell death. Importantly, IMP therapy was shown to have a positive effect on West Nile Virus (WNV) encephalitis, peritonitis, experimental autoimmune encephalomyelitis, post myocardial infarction cardiac function, renal reperfusion injury, and colitis. IMP provides an alternative, highly specific tool for inhibiting inflammatory monocytes in a MARCO-dependent manner. Due to the use of the natural leukocyte depletion pathway, IMP represents a novel and safe therapy specific for inflammatory monocytes.

In one aspect, the methods of the invention comprise inducing apoptosis of monocytes, granulocytes and/or neutrophils in the subject to reduce the severity of the inflammatory response or to reduce the duration of the inflammatory response. In one embodiment, administration of the negatively charged particles of the present invention induces apoptosis and clearance of monocytes, granulocytes and/or neutrophils, thereby aiding in the resolution of inflammation.

In one aspect, the methods of the present invention contemplate the use of the particles of the present invention as "molecular ravines" that bind to inflammatory molecules and polypeptides produced by cells, thereby preventing them from functioning. When inflammation occurs, cells such as macrophages and monocytes release pro-inflammatory mediators such as cytokines and chemokines into the surrounding pro-inflammatory environment. Examples of pro-inflammatory mediators include, but are not limited to, interleukins, TNF family members, interferons, and colony stimulating factors. These mediators enhance the inflammatory response, thereby exacerbating the inflammatory pathological state. As described herein, the particles of the invention bind to inflammatory mediators in the serum of animals that undergo an inflammatory immune response. Inflammatory mediators to which the particles of the invention are bound include, but are not limited to, heat shock protein beta-1, protein S100-A7, protein S100-A8, protein S100-A9, fatty acid binding proteins, annexin A1, and ubiquitin cross-reactive protein precursors. Administration of the uncoated particles of the invention to animals results in a reduction of inflammatory monocytes present in the inflammatory lesions, a reduction of inflammatory symptoms, and an increase in survival of the infected animals.

As discussed above, the present invention provides novel compounds having biological properties useful for treating immune-mediated disorders.

Thus, in another aspect of the invention, there is provided a pharmaceutical composition comprising particles and optionally a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In another embodiment, the particles of the invention may encapsulate or encapsulate an antigen. Alternatively, the particles of the present invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, the additional therapeutic agent for co-administration or inclusion in a pharmaceutical composition with a compound of the invention may be an approved anti-inflammatory agent, or it may be any of a variety of agents that are being approved at the food and drug administration (Food and Drug Administration) that are ultimately approved for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or bacterial or viral infection. It will also be appreciated that certain modified particles of the invention may exist in free form for use in therapy or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof.

The pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, as used herein, including any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, sixteenth Edition, e.w. martin (Mack Publishing co., easton, pa., 1980) ("lemngton pharmaceutical science, sixteenth edition, e.w. martin, microphone Publishing company, easton, pa, 1980) discloses various carriers for formulating pharmaceutical compositions, and known preparation techniques thereof. Unless any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effect, or otherwise interacting in a deleterious manner with any of the other components of the pharmaceutical composition, its use is contemplated as being within the scope of the present invention. Some examples of materials that may be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdery tragacanth; malt; gelatin; talc powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil and sesame oil; olive oil; corn oil and soybean oil; a diol; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and antioxidant agents, can also be present in the composition according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable formulations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed include water, ringer's solution, u.s.p. And isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-irritating, non-volatile oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

For example, the injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents, in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the action of a drug, it is often desirable to slow down the absorption of the drug by subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions or crystalline or amorphous materials which are poorly water soluble. The rate of absorption of a drug is thus dependent on its rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms is accomplished by dissolving or suspending the drug in an oily vehicle. The injectable depot forms are prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. In certain embodiments, drugs and therapeutic agents may be encapsulated in the particles of the invention for administration to a subject.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one of the following materials: inert pharmaceutically acceptable excipients or carriers, such as sodium citrate or dicalcium phosphate, and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) adsorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also have a composition such that they release the active ingredient(s) in a certain part of the intestinal tract, optionally in a delayed manner, only or preferentially. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

The modified particles may also be in microencapsulated form with one or more of the excipients described above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release control coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose and starch. Such dosage forms may in normal practice also contain other substances besides inert diluents, for example tabletting lubricants and other tabletting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also have a composition such that they release the modified particles in a certain part of the intestinal tract, optionally in a delayed manner, only or preferentially. Examples of embedding compositions that can be used include polymeric substances and waxes.

The invention encompasses pharmaceutically acceptable topical formulations of the modified particles of the invention. The term "pharmaceutically acceptable topical formulation" as used herein means any formulation that is pharmaceutically acceptable for intradermal administration of the modified particles of the invention by application of the formulation to the epidermis. In certain embodiments of the invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, polyols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., hypotonic or buffered saline) or any other carrier known in the art for topical administration of drugs. A more complete list of vectors known in the art is provided by standard references in the art, such as Remington's Pharmaceutical Sciences,16th Edition,1980and 17th Edition,1985 (Remington pharmaceutical sciences,16th edition,1980, and 17th edition, 1985), both of which are published by Mike publishing company (Mack Publishing Company, easton, pa.) of Iston, pa. The disclosure of which is incorporated herein by reference in its entirety. In certain other embodiments, the topical formulations of the present invention may comprise an excipient. Any pharmaceutically acceptable excipient known in the art may be used to prepare the pharmaceutically acceptable topical formulations of the present invention. Examples of excipients that may be included in the topical formulations of the present invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, other penetrating agents, skin protectants, surfactants, and propellants, and/or additional therapeutic agents used in combination with the modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulphite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents such as EDTA and citric acid. Suitable humectants include, but are not limited to, glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers for use in the present invention include, but are not limited to, citric acid buffers, hydrochloric acid buffers, and lactic acid buffers. Suitable solubilizing agents include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithins, and polysorbates. Suitable skin protectants that may be used in the topical formulations of the invention include, but are not limited to, vitamin E oil, allantoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topical formulations of the present invention comprise at least the modified particles of the present invention and a penetration enhancer. The choice of topical formulation will depend on several factors including the condition to be treated, the physicochemical characteristics of the compounds of the invention and other excipients present, their stability in the formulation, available manufacturing equipment, and cost constraints. As used herein, the term "permeation enhancer" means an agent that is capable of transporting a pharmacologically active compound through the stratum corneum and into the epidermis or dermis with preferably little or no systemic absorption. Various compounds have been evaluated for their effectiveness in enhancing the permeation rate of drugs through the skin. See, e.g., percutaneous Penetration Enhancers, maibach H.I.and Smith H.E. (eds.), CRC Press, inc. (eds.), boca Raton, fla (1995) ("transdermal permeation enhancer", maibach H.I. and Smith H.E. editions, CRC publishing company, bokapton, florida, 1995), which investigated the use and testing of different skin permeation enhancers, and Buyuktimkin et al, chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, gosh T.K., pfester W.R., yum S.I. (eds.), interph Press Inc., buffalo Grove, ill (1997) (Buyuktimkin et al, chemical means of transdermal drug permeation enhancement in transdermal and topical drug delivery systems, gosh T.K, pfeim W.R., editions, yum S.I., international publication, ill. Fowler, 1997). In certain exemplary embodiments, penetrants used in the present invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethanol, isopropyl alcohol, octylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decyl methyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methyl pyrrolidone.

In certain embodiments, the composition may be in the form of an ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant or patch. In certain exemplary embodiments, the formulation of the composition according to the present invention is a cream, which may further comprise saturated or unsaturated fatty acids, such as stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl alcohol or oleyl alcohol, stearic acid being particularly preferred. The cream of the present invention may also contain a nonionic surfactant, such as polyoxy-40-stearate. In certain embodiments, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers as may be required. Ophthalmic formulations, ear drops or eye drops are also contemplated as being within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches that have the added advantage of providing controlled delivery of compounds to the body. Such dosage forms are prepared by dissolving or dispersing the compound in a suitable medium. As discussed above, permeation enhancers may also be used to increase the flux of the compound through the skin. The rate may be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The modified particles may be administered by aerosol. This is achieved by preparing an aqueous aerosol, a liposome formulation or solid particles comprising the modified particles. Nonaqueous (e.g., fluorocarbon propellant) suspensions may be used.

Generally, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the agent with conventional pharmaceutically acceptable carriers and stabilizers. The carrier and stabilizer will vary depending on the requirements of the particular compound, but typically includes a nonionic surfactant (tween,Or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

It will also be appreciated that the modified particles and pharmaceutical compositions of the invention may be formulated and used in combination therapy, i.e., the compound or pharmaceutical composition may be formulated with or administered simultaneously with, before or after one or more other desired therapeutic agents or medical procedures. The specific combination of therapies (therapeutic agents or procedures) used in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies used may achieve a desired effect on the same disorder (e.g., the compounds of the invention may be administered simultaneously with another anti-inflammatory agent), or they may achieve different effects (e.g., controlling any adverse effects).

In certain embodiments, the pharmaceutical compositions comprising the modified particles of the present invention further comprise one or more additional therapeutically active ingredients (e.g., anti-inflammatory agents and/or palliative agents). For the purposes of the present invention, the term "palliative" refers to treatments that are focused on alleviating the symptoms of the disease and/or the side effects of the treatment regimen, but not curative. For example, palliative treatment encompasses analgesics, antihalations, and antiemetics.

The present invention provides a method of modulating an immune response in an individual (preferably a mammal, more preferably a human), the method comprising administering to the individual a modified particle as described herein. The immunomodulation methods provided by the present invention include methods of suppressing and/or inhibiting an innate immune response or an adaptive immune response, including, but not limited to, an immune response stimulated by an immunostimulatory polypeptide or viral or bacterial component.

The modified particles are administered in an amount sufficient to modulate the immune response. Modulation of the immune response may be humoral and/or cellular, as described herein, and measured using standard techniques in the art and as described herein.

In certain embodiments, the subject suffers from disorders associated with unwanted immune activation, such as allergic diseases or conditions, allergies, and asthma. An individual with an allergic disease or asthma is an individual with identifiable symptoms of an existing allergic disease or asthma.

In certain embodiments, the individual suffers from disorders associated with unwanted immune activation, such as autoimmune diseases and inflammatory diseases. An individual with an autoimmune or inflammatory disease is an individual with identifiable symptoms of the existing autoimmune or inflammatory disease.

Autoimmune diseases can be divided into two broad categories: organ specificity and systemic. Autoimmune diseases include, but are not limited to, rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), type I diabetes, type II diabetes, multiple Sclerosis (MS), immune-mediated infertility such as premature ovarian failure, scleroderma, sjogren's disease, vitiligo, alopecia (baldness), multiple gland failure, graves' disease, hypothyroidism, polymyositis, pemphigus vulgaris, pemphigus largehead, inflammatory bowel diseases (including crohn's disease and ulcerative colitis), celiac disease, autoimmune hepatitis (including hepatitis associated with Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV), hypopituitary disease, graft versus host disease (GvHD), myocarditis, addison's disease, autoimmune skin disease, uveitis, pernicious anemia, and hypoparathyroidism.

Autoimmune diseases may also include but are not limited to, amyotrophic Lateral Sclerosis (ALS), hashimoto thyroiditis, autoimmune polyadenylic syndrome types I and II, paraneoplastic pemphigus, vesicular pemphigoid, dermatitis herpetiformis, linear IgA disease, acquired epidermolysis bullosa, erythema nodosum, gestational pemphigoid, cicatricial pemphigoid, primary mixed cryoglobulinemia, pediatric chronic bullous disease, hemolytic anemia, thrombocytopenic purpura, godpasture's syndrome, autoimmune neutropenia, myasthenia gravis, eton-Rabbet's muscle weakness syndrome, stiff man syndrome, acute disseminated encephalomyelitis, grin-Barli syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy with conduction block, chronic neuropathy with monoclonal gammaglobulopathy, strabismus-myoclonus syndrome, cerebellar degeneration, encephalomyelitis, retinopathy, primary biliary sclerosis, sclerosing cholangitis, gluten-sensitive enteropathy, ankylosing spondylitis, reactive arthritis, polymyositis/dermatomyositis, mixed connective tissue disease, behcet's syndrome, psoriasis, polyarteritis nodosa, allergic vasculitis and granuloma (Churg-Strauss disease), polyangitis overlap syndrome, hypersensitivity vasculitis, wegener's granulomatosis, temporal arteritis, large arteritis, kawasaki disease, isolated vasculitis of the central nervous system, thromboangiitis obliterans, sarcoidosis, glomerulonephritis and cold disease. These conditions are well known in the medical arts and are described, for example, in Harrison' S Principles ofInternal Medicine,14th ed., fauci A S et al, eds., new York: mcGraw-Hill,1998 (Harrison Endoconcha., 14th edition, fauci A S et al, new York, maglahal, 1998).

The subject treated by the particles of the invention is preferably a human, however, the particles may be used to treat non-human animal species. Non-human animal species that may be treated by the particles of the present invention include, but are not limited to, dogs, cats, chickens, geese, ducks, sheep, cows, goats, pigs, non-human primates, monkeys, rabbits, mice, rats, guinea pigs, hamsters, gerbils, and horses.

Animal models for studying autoimmune diseases are known in the art. For example, animal models that exhibit the most similarity to human autoimmune diseases include animal lines that spontaneously develop a high incidence of the particular disease. Examples of such models include, but are not limited to, non-obese diabetic (NOD) mice that develop diabetes-like 1 disease and animals that are prone to lupus-like disease, such as New Zealand hybrids, MRL-Fas ^lpr BXSB mice. Animal models that have induced autoimmune diseases include, but are not limited to, experimental Autoimmune Encephalomyelitis (EAE) as a model of multiple sclerosis, collagen-induced arthritis (CIA) as a model of rheumatoid arthritis, and Experimental Autoimmune Uveitis (EAU) as a model of uveitis. Animal models of autoimmune diseases have also been constructed by genetic manipulation and include, for example, IL-2/IL-10 gene knockout mice for inflammatory bowel disease, fas or Fas ligand gene knockout for SLE, and IL-I receptor antagonist gene knockout for rheumatoid arthritis.

In certain embodiments, the subject is suffering from a bacterial or viral infection. An individual with a bacterial or viral infection is an individual with identifiable symptoms of an existing bacterial or viral infection.

A non-limiting list of viral infections treatable with the modified particles of the invention include herpes virus infections, hepatitis virus infections, west nile virus infections, flavivirus infections, influenza virus infections, rhinovirus infections, papillomavirus infections, paramyxovirus infections, parainfluenza virus infections, and retrovirus infections. Preferred viruses are those that infect the central nervous system of a subject. Most preferred viruses are those that cause encephalitis or meningitis.

A non-limiting list of bacterial infections treatable with the modified particles of the invention include staphylococcal infections, streptococcal infections, mycobacterial infections, bacillus infections, salmonella infections, vibrio infections, spirochete infections, and Neisseria infections. Preferred are bacteria that infect the central nervous system of the subject. Most preferred are bacteria that cause encephalitis or meningitis.

In some embodiments, the invention relates to the use of the compositions of the invention prior to onset of disease. In other embodiments, the invention relates to the use of the compositions of the invention to inhibit developing disease. In some embodiments, the invention relates to ameliorating a disease in a subject. By ameliorating a disease in a subject is meant to include treating, preventing or inhibiting a disease in a subject.

In some embodiments, the invention relates to preventing recurrence of a disease. For example, an unwanted immune response may occur in a region of the peptide (such as an antigenic determinant). Recurrence of diseases associated with unwanted immune responses may occur as a result of immune response attacks on different regions of the peptide. Since the negatively charged particles of the present invention do not contain attached peptide or antigen moieties, the particles will be effective against multiple epitopes. T cell responses in some immune response disorders, including MS and other ThI/17 mediated autoimmune diseases, can be dynamic and evolve during relapsing remitting and/or chronic progressive disease. The dynamic nature of T cell repertoires suggests that it may be useful to treat certain diseases, as targets may change as the disease progresses. Prior to this, it was necessary to know the reaction type in advance in order to predict the progression of the disease. The present invention provides a composition that can prevent the action of a dynamically changing disease, i.e., the function of "epitope spreading". A known model for relapse is the immune response to proteolipoproteins (PLP) as a model for Multiple Sclerosis (MS). The initial immune response may occur through a reaction to PLP 139-15. Subsequent episodes of the disease can occur by recurrent immune responses to PLP [ pi ] s-iβi.

Certain embodiments of the invention relate to the treatment of pathological conditions associated with unwanted hypersensitivity reactions. The hypersensitivity reaction may be any of types I, II, III and IV. Immediate type (type I) hypersensitivity. The frequency of administration generally corresponds to the timing of allergen exposure. Suitable animal models are known in the art (e.g., gundel et al, am. Rev. Respir. Dis. 1:46:369, 1992 (Gundel et al, U.S. respiratory disease theory, 146, page 369, 1992), wada et al, J. Med. Chem.39,2055,1996 (Wada et al, journal of pharmaceutical chemistry, 39, page 2055, 1996), and WO 96/35418).

Other embodiments of the invention relate to transplantation. This refers to the transfer of a tissue sample or graft from a donor individual to a recipient individual, and is often performed on a human recipient who requires tissue in order to restore the physiological function provided by the tissue. Transplanted tissue includes, but is not limited to, whole organs such as kidney, liver, heart, lung; organ components such as skin grafts and cornea; and cell suspensions such as bone marrow cells and cell cultures selected and expanded from bone marrow or circulating blood, as well as whole blood transfusion.

Any serious potential complications of transplantation will occur due to antigenic differences between the host recipient and the transplanted tissue. Depending on the nature and extent of the differences, there may be a risk of immune attack by the host against the graft or against the host by the graft, or both. The extent of risk is determined by tracking the response patterns in a population of similarly treated subjects with similar phenotypes, and correlating various possible contributors according to accepted clinical procedures. The immune attack may be the result of a pre-existing immune response (such as a pre-formed antibody) or an immune response initiated at about the time of implantation (such as the generation of TH cells). Antibodies, TH cells, or Tc cells may be involved in any combination with each other and with various effector molecules and cells. However, antigens involved in immune responses are generally unknown and therefore pose difficulties in designing antigen-specific therapies or inducing antigen-specific tolerance. The modified particles of the invention are particularly useful for preventing organ rejection because no attached peptide or antigen is required to conjugate the modified particles to render the particles effective in inducing tolerance or ameliorating inflammatory immune responses.

Certain embodiments of the invention relate to reducing host versus graft disease, thereby causing a risk of rejection of the tissue graft by the recipient. Treatment may be administered to prevent or reduce the effects of hyperacute rejection, acute rejection or chronic rejection. Preferably, treatment is initiated long enough prior to implantation so that tolerance is in place upon placement of the implant; but in cases where such is not possible, treatment may be initiated simultaneously with or after the implantation. Regardless of the time of onset, treatment will generally last periodically for at least the first month after implantation. Subsequent doses may not be required if adequate adaptation of the graft occurs, but may be restarted if there is any sign of graft rejection or inflammation. Of course, the tolerizing procedure of the present invention may be combined with other forms of immunosuppression to achieve even lower levels of risk.

Certain embodiments of the invention relate to alleviating or otherwise ameliorating an inflammatory response induced as a response to surgery. In one embodiment of the invention, the immunomodifying particles are administered prior to surgery. In another embodiment of the invention, the immunomodifying particle is administered simultaneously with or during surgery. In yet another embodiment of the invention, the immunomodifying particle is administered after surgery.

The particles of the invention may also be used to treat abscesses or purulence to alleviate inflammatory responses in subjects following exposure to infectious pathogens such as bacteria or parasites. In one embodiment of the invention, the immunomodifying particles are administered in combination with antibacterial and/or antiparasitic therapies as known in the art.

The particles of the present invention may also be used to reduce or otherwise ameliorate inflammatory responses induced as a response to physical wounds or lesions, including, but not limited to, sports injuries, wounds, spinal cord injuries, brain injuries, and/or soft tissue injuries. In one embodiment of the invention, the immunomodifying particle is administered after the subject has experienced a wound or injury.

The particles of the invention may also be used to reduce inflammatory responses associated with the development and/or growth of cancer cells. Cancers that may be treated include, but are not limited to, central nervous system cancers, basal cell cancers, cancerous brain tumors, burkitt's lymphoma, lymphomas, cervical cancer, ovarian cancer, testicular cancer, liver cancer, non-small cell and small cell lung cancer, melanoma, bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, renal (renal cell) cancer, leukemia, non-hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer. In one embodiment, subcutaneous injection of the particles of the present invention prevents accumulation of inhibitory neutrophils or monocyte-derived suppressor cells, thereby enhancing or promoting a tumor-specific immune response in a cancer patient.

The particles of the invention may also be used for regeneration of damaged tissue. In one embodiment, administration of the particles to a patient enhances regeneration of damaged epithelial cells in the digestive tract. In another embodiment, the patient is suffering from ulcerative colitis, crohn's disease, or inflammatory bowel disease. In another embodiment, administration of the particles of the invention to a patient enhances remyelination of neurons. In another embodiment, the patient is suffering from multiple sclerosis.

Examples

The following examples are provided to further illustrate the advantages and features of the present invention and are not intended to limit the scope of the present disclosure.

Materials and methods

A mouse

For WNV and IBD studies, eight week old female C57BL/6 mice were obtained from the animal resource center (Animal Resources Centre (WA, australia)) in Western Australia, australia. All procedures were carried out under the permission of the university of Sydney animal ethics Committee (University of Sydney Animal Ethics Committee). For EAE studies, female SJL/J mice were obtained eight weeks old from haren laboratories (Harlan Laboratories (Indianapolis, IN USA)) of Indianapolis, indiana. For thioglycolate studies, eight week old females Balb/c were obtained from the national cancer institute (National Cancer Institute (MD, USA)) in maryland, USA. MARCO-/-animals with Balb/c background were offered by Lester Kobzik friends, university of Harvard, massachusetts, USA (Harvard University, MA). For cardiac inflammation studies, twelve week old male C57BL/6 mice were purchased from Jackson laboratories (Jackson Laboratory (ME, USA)) in Burmese, U.S.A. All procedures were performed under the permission of the university of northwest animal administration and use committee (Northwestern University Institutional Animal Care and Use Committee). All animals were housed under specific pathogen-free conditions, with food and water ad libitum.

WNV infection

WNV (Sarafend strain) originates from the brain of neonatal mice and is propagated in vitro cell culture (Getts et al, J. Exp Med.205:2319-2337,2008 (Getts et al, journal of Experimental medicine, volume 205, pages 2319-2337, 2008)). WNV infection was performed in C57BL/6 animals (Getts et al, J.Exp Med.205:2319-2337,2008 (Getts et al, journal of laboratory medicine, 205, pages 2319-2337, 2008)). Mice were infected intranasally with 6X 104 or 6X 103PFU WNV in 10. Mu.L of sterile phosphate buffered saline (PBS; invitrogen, calif., USA) of Engineers, calif. Only sterile PBS was used to simulate 24 infection. Mice were weighed daily after infection.

Thioglycolate induced peritonitis

Induction of peritonitis was performed by intraperitoneal injection of 1ml of 4% (w/v) thioglycolate broth prepared in sterile water (Sigma Aldrich, MO, USA, sigma Aldrich, USA). The leukocytes were isolated at defined time points by peritoneal lavage (washing) using ice-cold 0.05mM EDTA PBS solution.

EAE induction

PLP139-151 (HSLGKWLGHPDKF) peptide-induced EAE was induced in SJL/J mice (Getts et al, J.Immunol.187:2405-2417,2011 (Getts et al, J.Immunol.187, volume 187, pages 2405-2417, 2011)). Individual animals were observed daily and clinical scores were assessed on the following scale 0-5 in a blind-check format. 0 = no anomaly; 1 = tail weakness or hind limb weakness; 2 = tail weakness and hind limb weakness; 3 = hind limb paralysis; 4 = hind limb paralysis and forelimb weakness; and 5 = dying state. These data are recorded as average clinical scores. The paralyzed animals can obtain food and water more easily.

Myocardial infarction

Myocardial infarction was performed in C57BL/6 mice. The left anterior descending branch artery was permanently occluded by surgery (Yeap et al Methods In Molecular Biology, in Press (Yeap et al, methods of molecular biology, publications)).

Renal ischemia reperfusion induction

The renal arteries were ligated for 45 minutes and then released to allow complete reperfusion of the kidneys.

Inflammatory bowel disease induction

DSS-induced colitis model to induce IBD (Bao et al, immunol. Cell bio.89:853-860,2011 (Bao et al, immunology and cytobiology, volume 89, pages 853-860, 2011)). 2.5% w/v 25 dextran sodium sulfate (DSS, MW 36,000-44,000D; ICN biomedical Co., australia, francia) dissolved in tap water was ad libitum administered for 9 consecutive days 41. During this period the control group received only tap water. Body weight and clinical assessment were measured daily. Percent weight loss was calculated as follows: ((average body weight at day 0-9/body weight at day 0). Times.100). After weight measurement, mice were examined for clinical parameters including mobility and gait, voice, group interaction and grooming behavior. Each parameter was assigned a score of 0-2, where the total number of 0-1 represents normal, 2-4 mild, 5-7 moderate, 8-10 severe 41. The evaluation value of excrement was measured as daily as possible. Scoring is given to fecal consistency, hematochezia, and rectal bleeding. Total values between 0 and 6 reflect the fecal score, with a value of 0 representing normal appearance of fecal matter and a value of 6 representing severe diarrhea, rectal bleeding and bloody stool.

Intravenous delivery of particles

FITC or Brilliant Blue (BB) floresbrite common and carboxylated polystyrene particles (0.5 μm diameter) were obtained from Polysciences corporation of PA, USA. FITC poly (lactic-co-glycolic acid) (PLGA) and FITC polystyrene plain, carboxylated and aminated particles (500 nm diameter) were obtained from the Phosphorex company of MA, USA. Carboxylated 500nm nanodiamonds were produced at the university of mecaremia (Macquarie University) or the university of sydney (Sydney University) in new south wilfory, australia. The particles were diluted to the indicated concentration in sterile PBS and, as indicated, 200 μl was injected intravenously. Vehicle control animals received only 200 μl sterile PBS. Tissues are harvested at designated time points and processed for flow cytometry, histology, or immunohistochemistry. Ly6C from bone marrow was performed as described in Getts et al 2008 ^hi Isolation and sorting of inflammatory monocytes wherein isolated cells were labeled with PKH26 (Invitrogen) and injected intravenously at day 6 post-immunization with 2.0X10 s in recipients mimicking infection and WNV infection ⁶ Individual cells. Specific particle properties are defined in table 3.

Flow cytometry

Mice were anesthetized and perfused with 50mL sterile PBS. Spleen, brain, bone marrow, blood and peritoneal fluid were isolated and processed into single cell suspensions (Getts et al, j.exp med.205:2319-2337,2008 (Getts et al, journal of experimental medicine, volume 205, pages 2319-2337, 2008)). Cells were incubated with anti-CD 16/32 and viable cells, which typically showed >95% viability, were counted using trypan blue exclusion experiments. The cells were then incubated with fluorescent-labeled antibodies (Biolegend) against CD45, CD11b, ly6C, ly6G, CD c, CD103, ETC. Cell surface molecule expression was measured on FACS LSR-II using the FACS Diva program (Becton Dickinson, NJ, USA) in new jersey. The living cell population is gated by forward and side scatter, and the identified fluorescent cell population is then assayed by forward gating. The acquired FACS data file was analyzed using a Flow Jo (Tree Star, inc. Or, USA) program of Flow Jo (oregon, USA). Quantification of the cell population of interest was calculated based on the percentage flow cytometer at the time of analysis and the absolute cell count from each organ.

Adoptive transfer

On day 6 post immunization, bone marrow of WNV infected animals was treated as a single cell suspension and incubated with fluorescent-labeled antibodies to CD11b, ly6C, and Ly 6G. CD11b+, ly6Chi, ly 6G-monocytes were sorted on FACS Aria using the FACS Diva program (Becton Dickinson), with the stringency set to achieve >98% purity. Cells were then labeled with the fluorescent membrane dye PKH26 (Invitrogen) according to the manufacturer's instructions. On day 6 post immunization, 2.0X106 sorted Ly6Chi monocytes were injected intravenously for matched recipients of the mock and WNV infection and delivered in 200. Mu.L PBS. Brain and spleen were isolated from recipients on day 7 post immunization (24 hours post transfer) and treated for flow cytometry as described above.

Histology and immunohistochemistry

Mice were anesthetized and perfused with 50mL sterile PBS. Except for the heart (hearts were processed into paraffin blocks) (Getts et al, j. Neurochem 103:10919-1030,2007 (Getts et al, journal of neurochemistry, volume 103, pages 10919-11030, 2007)), all organs were isolated and snap frozen in optimal cutting temperature compounds (Optimum Cutting Temperature Compound) (OCT; tissue-Tek, tokyo, japan) cut into eight micrometer Tissue slices in a cryostat, air-dried overnight, then stored at-80 ℃ until needed, frozen sections were thawed and histologically (standard hematoxylin-eosin staining) or immunohistochemistry (Getts et al, j. Exp Med 205:2319-2337,2008 (Getts et al, journal of experimental medicine, volume 205, pages 2319-2337, 2008)) using images for MARCO, signa-R1 and siju 1 (DP), and figure 1, figure 2, tsujin, 35, b.35, and image (35 b.m) were acquired using the microscope software (figure 35, MA, 35, and mg, b.p.m.p.m. 35, j.p.m., as indicated).

Histology and immunohistochemistry

Multiplex ELISA

A multiplex plate ELISA (Quansys Biosciences Co., ltd. (Quansys Biosciences)) was performed using the Quansys Q-plex mouse cytokine Screen IR 16-plex according to the manufacturer's instructions. The plate was observed on a Li-Cor Odyssey IR imaging system (Li-Cor Biotechnology Co., ltd. (Li-Cor Biotechnology)). Images were analyzed using the Quansys Q-view software (Quansys biological science Co., ltd. (Quansys Biosciences)).

Table 3: physical Properties of the particlesPhysical Properties

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Statistics of

Charts were made in GraphPad Prism (GraphPad software company (GraphPad software, SDG, USA) of san diego, USA) and statistical analysis was performed. To compare two samples, unpaired two-tailed student T-test was performed. To compare three or more samples, a one-way anova with Tukey-Kramer post test was performed. For survival data, a Mantel-Haenszel log rank test was performed. For these tests, p.ltoreq.0.05 (x) was considered significant, and p.ltoreq.0.01 (x) was considered very significant.

Example 1

Conditioned IMP has reduced ability to inhibit macrophage migration to the brain。

Day 7 plasma was removed from mock or WNV infected animals and incubated with PS-IMP. On day 6 post immunization, these "conditioned" particles or unconditioned (untreated microbead) particles were reinfused into WNV infected mice. On day 7, inflammatory monocyte influx in the brain was examined by flow cytometry. Although untreated (normal IMP) resulted in reduced monocyte influx in the brain, the mock plasma coated IMP failed to inhibit monocyte migration (fig. 9). WNV serum coated IMP has a reduced ability to inhibit monocytes relative to uncoated IMP, but is still able to reduce migration to some extent.

Example 2

Immune-modified particles reduce inflammatory monocyte migration and improve WNV survivalRate of

The hallmark of acute and many chronic inflammatory diseases is the influx of monocytes into the inflammatory area. Previously, we have demonstrated that the pathogenesis associated with WNV brain infection is a direct consequence of inflammatory monocyte trafficking into the brain (Getts, 2008; getts,2007; terry,2012; getts, 2012). In this model, 5% weight loss is a biomarker associated with the transport of Φim into the brain and therefore mortality (Getts et al, j. Neurooin-fermentation, in Press (Getts et al, journal of neuroinflammation, publication)). At least 50% of infected animals will continue to lose weight, with 100% generally dying from infection within 72 hours of initial weight loss (fig. 1A). Animals that did not lose weight did not show any other symptoms of encephalitis, but still developed a lifelong clear immunity (fig. 1A). To monitor monocyte transport, carboxylated, negatively charged polystyrene particles (PS-IMP) were unexpectedly infused instead of polystyrene neutral particles (PS-NP) at initial weight loss (day 6). Surprisingly, infusion almost immediately reduced symptoms such as coat wrinkling, discomfort and seizures, which are consistently associated with clinical WNV Central Nervous System (CNS) infections. This reduction was associated with a reduction in local pro-inflammatory cytokines and chemokines (fig. 6). Furthermore, daily infusions of PS-IMP for up to 5 days resulted in 60% survival of mice that would otherwise die from infection (fig. 1A). In these mice, body weight was generally stable and returned to control levels within 5-6 days (not shown). PS-NP or vehicle treated mice showed sustained weight loss with a survival rate of 10% or less. In addition, pre-conditioning of IMP with serum from WNV or mock-infected animals prior to infusion did not affect disease prognosis (not shown). Consistent with our previous observations in WNV encephalitis (Getts, 2008; getts,2007; terry,2012; getts, 2012), survival correlated with a significant reduction in Φim-derived macrophages in the brain of PS-IMP treated mice compared to phosphate buffered saline (vehicle) -treated or PS-NP-treated control animals (fig. 1B, fig. 7).

Example 3

The immunomodifying particles must be negatively charged

Treatment of WNV infected animals with other carboxylated particles (specifically ND IMP and PLGA-IMP) resulted in survival statistics similar to those seen in animals treated with PS-IMP (fig. 1C), indicating that the particle core was independent of the nature responsible for the therapeutic effect. Initial experimental studies used carboxylated PS-IMP with zeta potential less than-50 mV (table 3). We therefore compared PS particles having a zeta potential of-50 mV, PS particles having a zeta potential of about-0.5 mV (neutral; polystyrene neutral particles; PS-NP) or PS particles having a zeta potential of +40mV (aminated particles, polystyrene positive particles; PS-PP). PS-IMP (-50 mV) showed the greatest effect in reducing inflammatory monocyte trafficking to the brain (FIG. 1D). In contrast, PP did not reduce infiltration of IM-derived macrophages (fig. 1D), nor did PP improve survival (fig. 1E). Dose response of 500nm PS-IMP in mice with WNV encephalitis showed a maximum effective dose of about 4x109 IMP particles or 0.355mg particles/mouse (fig. 1F-G).

Example 4

PS-IMP treatment resulted in redirection of inflammatory macrophages to the spleen

After infusion, PS-IMP was localized mainly to the lung, spleen and liver (FIG. 2A), and no FITC was present in the brain and thymus ⁺ Particles (fig. 7A-B) and no particles were present in the peripheral lymph nodes (data not shown). Flow cytometry revealed that IMP was found in inflammatory monocytes, BB20 ⁺ B cell, CD3 ⁺ Relatively uniform distribution between T cells and NK1.1 cells (fig. 2B-C), however, in infected mice, significantly more IMP was found to be taken up by inflammatory monocytes (fig. 2B-C) than by any other cell type in the spleen in the blood circulation, mimicking the phenotype of IMP-containing monocytes in infected mice as Ly6C negative, whereas in infected animals IMP was predominantly localized to Ly6C positive monocytes (fig. 2D-E). Furthermore, spleens from WNV-infected mice treated with FITC-PS-IMP had significantly more inflammatory monocytes (FIG. 2F; FIG. 9) than mice treated with NP or vehicle control, closely corresponding to the reduction of circulating inflammatory monocytes in the peripheral blood of these mice (FIG. 2G).

It has been previously shown that the onset of WNV encephalitis is due to inflammatory mononuclear finenessCell induction (Terry et al 2012; getts et al 2012). When combining these previous findings with the data obtained here, we speculate that IMP mediates its therapeutic activity by: binding to inflammatory monocytes eliminates their migration to the inflamed brain, and instead causes their migration to the spleen. To confirm this, ly6C was sorted from bone marrow of WNV infected mice on day 6 post immunization ^hi Monocytes were labeled with PKH26 and were intravenously transferred to recipients mimicking infection and WNV infection on day 6 post-immunization (fig. 3A). Then only PS-IMP, NP or vehicle was injected. PKH 26-labeled Ly6C ^hi Inflammatory monocytes trafficked into WNV-infected brains, differentiated into macrophages, and no peripheral immune cells were observed in mock-infected brains (fig. 3B, data not shown). As described by Getts et al (J. Exp. Med.205:2319-2337,2008 (journal of experimental medicine, vol. 205, pages 2319-2337, 2008)), PS-IMP treatment reduced infiltration of host inflammatory monocytes into WNV-infected brain (FIG. 3B), and significantly fewer adoptive transfer of PKH26 ⁺ Cells migrate into the brain (fig. 3B and 3D). PKH 26-labeled cell migration into the spleen was observed in mock-infected and WNV-infected mice (FIGS. 3C and 3E), however, PS-IMP treatment resulted in significantly more Ly6C in the spleen of WNV-infected mice ^hi Monocyte accumulation (fig. 3E). The importance of spleen in the efficacy of IMP treatment was demonstrated in splenic mice. IMP infusion failed to reduce migration of inflammatory monocytes into the brain of WNV-infected splenic mice compared to splenic, vehicle-treated, WNV-infected control mice (fig. 3F). Recently it has been demonstrated that the spleen has a reservoir of monocytes identified as CD11b ⁺ /Ly6C ^hi /CD11c ^- They can recruit from the spleen under certain inflammatory conditions (Swirski et al, science 325:612-616;2009 (Swirski et al, science 325, pages 612-616, 2009); leuschner et al, J.exp. Med.209:123-137,2012 (Leuschner et al, journal of experimental medicine 209, pages 123-137, 2012); robbins et al, circulation 125:364-374,2012 (Robbins et al, circulation 125, volumes 364-374, 2012)). We speculate that IMP infusion enhanced this spleen mononuclear cell pool. However, ingestIMP and migration to more than 70% of the splenic adoptively transferred monocytes expressed CD11c and CD103 (fig. 3E and 3F), making this speculation unlikely. Taken together, these data indicate that infused IMP is taken up by inflammatory monocytes, which are transferred to the spleen, resulting in a reduced number of inflammatory monocytes in the blood available for migration to the site of inflammation.

Example 5

IMP inhibits migration of inflammatory monocytes into inflamed peritoneum

It is apparent that monocytes are important mediators of the immunopathological state observed during WNV encephalitis, however, infection results in a complex series of immune responses involving other cell subsets. To address the specificity of IMP for inflammatory monocytes during inflammation, a model of aseptic macrophage-mediated peritoneal inflammation was used. Migration of leukocytes into the thioglycolate pro-inflammatory peritoneum follows a conventional pattern in which neutrophils are triggered within the first 4-18 hours, followed by CCR2 dependent accumulation of Φim-derived macrophages from about 12 hours (Tsou et al, 2007). Infusion of PS-IMP (fig. 7D-E; fig. 4A-B) or PLGA-IMP (not shown) 24 hours after intraperitoneal thioglycolate injection significantly reduced the transport of inflammatory monocytes to the site of inflammation, which was eliminated in splenic mice (fig. 7F). Together, this data highlights not only the anti-inflammatory properties of IMP in the primary macrophage-mediated inflammatory model, but also the importance of spleen to IMP efficacy.

Example 6

Inflammatory monocytes express MARCO, which is critical for IMP activity。

Particles such as IMP will be taken up through the scavenger receptor pathway (Kanno et al, 2007). One key scavenger receptor specifically involved in binding negatively charged particles as well as polystyrene is MARCO (Chao et al 2012; kanno et al 2007). In the spleen, PS-IMP co-localizes in the border region with MARCO-expressing cell populations, similar to what we previously described for apoptotic cell uptake following intravenous infusion (Getts et al, 2011; getts et al, 2012) (FIG. 4C).It was also found that Ly6c isolated from the spleen of WNV infected animals (rather than mock infected animals) ^hi /CD11b ⁺ /CD11c ^- On Φim, MARCO was up-regulated (fig. 4D). To further illustrate the effect of MARCO, thioglycolate was used in MARCO-deficient (MARCO ^-/- ) Peritoneal inflammation was induced in animals. 48 hours after thioglycolate administration, from wild-type mice and MARCO ^-/- A similar number of peritoneal monocytes were isolated from mice (fig. 4B, 4E). However, PS-IMP treatment resulted in a reduced number of Ly6C in peritoneum in wild type mice ^hi /CD11b ⁺ PhiIM, unlike wild-type mice, PS-IMP treatment infusion did not reduce slave MARCO ^-/- Animal isolated peritoneal Ly6C ^hi The number of/CD 11B macrophages (FIGS. 4B, 4D), which directly indicate the effect of MARCO on the uptake and therapeutic efficacy of IMP. In addition, MARCO has been shown to play a direct role in apoptosis induction in macrophages phagocytosed with silica particles (Hamilton et al, 2006). Interestingly, at 2 hours after PS-IMP (FIGS. 4G-I; FIG. 11B) or PLGA-IMP (not shown) infusion, IMP significantly increased wild type mice (rather than MARCO ^-/- Mice) number of annexin V and caspase-3 positive inflammatory monocytes in the spleen. Apoptosis was induced in both neutrophils and inflammatory monocytes and was detectable within 2 hours after IMP infusion (fig. 10). Mice were injected with PS-IMP or PBS and sacrificed at the subsequent 2 hours. The spleens were removed, treated to single cell suspensions, and stained with anti-CD 11b, ly6C, CD b, ly6G, fixable cell viability dye (Fixable viability dye) eFluor780 (eBioscience). Using Caspglow ^TM Apoptotic cells were detected using a fluorescein-activated caspase-3Staining Kit (Fluorescein Active Caspase-3 labeling Kit). Infusion of PLGA-IMP into thioglycolate-induced wild-type mice resulted in Ly6C expressing the apoptosis markers annexin V and caspase-3 ^hi Phi IM and Ly6G ⁺ The number of neutrophils increases significantly. This was not observed in the mock treated animals. Two hours after injection (FIG. 11), PS-IMP was localized to CD11b+Ly6C+Ly6G-monocytes in spleens of mock and TG peritonitis-induced mice. Ingestion and finger of PS-IMPThe activity of the enzyme caspase-3, which is shown to be apoptotic, is related (a). In PS-IMP treated, TG peritonitis induced mice, an increased number of monocytes were positive for caspase-3 activity (b).

Very few (less than 5%) necrotic cells, indicating that cell death is not associated with degradation of IMP.

Taken together, the data indicate that IMP may be taken up by MARCO scavenger receptors, which mediate the downstream signaling 7 pathway that leads to inflammatory monocyte migration, accumulation and subsequent apoptosis in the spleen.

Example 7

IMP therapy inhibits inflammatory monocytes in EAE

To further understand the therapeutic potential of IMP, a number of unrelated inflammatory disease models were examined. First, another non-infectious model of CNS inflammation, experimental Autoimmune Encephalomyelitis (EAE), was tested. In EAE, recycled Ly6C ^hi Inflammatory monocytes differentiate into macrophages and microglia, in EAE these same cells differentiate predominantly into CD11c which promotes T cell activation and epitope expansion ⁺ DC (Getts et al, J.Exp. Med.205:2319-2337,2007 (Getts et al, vol. 205, pp. 2319-2337, 2007)), king et al, blood 113:3190-3197,2009 (King et al, blood, pp. 113, 3190-3197, 2009), getts et al, J.Neurol flash, in Press (Getts et al, J.neuroinflammation, publication)). Intravenous infusion of biodegradable PLGA-IMP for 7 days per day at the onset of disease not only ameliorates the disease during treatment, but also correlates with prolonged efficacy as determined by the disappearance of symptoms 14 days after cessation of treatment (fig. 5A). More importantly, the daily infusion of IMP treatment during primary disease resulted in decreased disease scores and inhibition of recurrence initiation during the treatment period (fig. 5B). Reduced disease scores in animals treated at the time of onset correlated with reduced inflammation in the spinal cord as determined by flow cytometry (fig. 5C, D and fig. 8A), and the most significant reductions were observed in the DC compartments of inflammatory monocyte origin (fig. 5C-D and fig. 8A)8A) A. The invention relates to a method for producing a fibre-reinforced plastic composite Similar to observations in animals afflicted with WNV encephalitis or peritonitis, the reduction of inflammatory monocytes in EAE CNS was associated with significant accumulation of Ly6C expressing monocytes in the spleen (fig. 5D and 8B).

Example 8

IMP therapy reduces damage caused by ischemia reperfusion

Inflammatory monocytes are involved in the onset of heart diseases including atherosclerosis and myocardial infarction and are associated with poor prognosis (Swirski et al, science 325:612-616,02009 (Swirski et al, science, volume 325, pages 612-616, 2009); leuschner et al, J.exp. Med.209:123-137,2012 (Leuschner et al, journal of experimental medicine, volume 209, pages 123-137, 2012); robbins et al, circulation125:364-374,2012 (Robbins et al, circulation125, pages 364-374, 2012); bailey et al, nat. Immunol 8:172-180,2007 (Bailey et al, nature immunology, volume 8, pages 172-180, 2007)). The effect of PLGA-IMP treatment for 3 days was determined using a permanent left anterior descending arterial occlusion model (Yeap et al Methods In Molecular Biology, in Press (Yeap et al, methods of molecular biology, publications)). In vehicle treated animals, occlusion resulted in strong infiltration of monocytes into the myocardium, involving up to 40% of the left ventricle wall (fig. 5I-J). IMP treatment significantly reduced the size of the inflammatory lesions, reducing overall cardiac inflammation by 15-20% (fig. 5i, j). In addition, CD68 in the infarct zone was observed ⁺ The number of macrophages was significantly reduced and IMP treatment resulted in CD68 relative to vehicle-treated controls ⁺ Cell/mm ² Is reduced by 30% (fig. 5K, L). Importantly, this decrease is also similar to Ly6C in the spleen ^hi A significant increase in the number of inflammatory monocytes correlates (fig. 8D-E). To further illustrate the potential of IMP therapy in reperfusion injury models, IMP was tested in rodents, where the renal artery was ligated for 45 minutes, followed by return of blood flow. IMP treatment was started 12 hours after ligation. Notably, serum creatinine was cleared on day 1 and day 5 in animals that had been treated with IMP compared to vehicle-treated controlsSignificantly larger (n) associated with reduced tubular atrophy scores in animals treated at these time points (o). In summary, it has been well established that graft failure during transplantation and long-term prognosis after myocardial infarction are directly related to the extent of inflammation and infarct size, respectively (Nahrendorf et al, circulation 121:2437-2445,2010 (Nahrendorf et al, circulation 121, vol. 2437-2445, 2010); leuschner et al, J. Exp. Med.209:123-137,2012 (Leuschner et al, vol. 209, 123-137, 2012); nahrendorf et al, J. Exp. Med.204:3037-3047, 2007), J. Exp. Med.204:3037-3047, 2007). The data herein indicate that IMP is strongly supported not only in terms of reducing inflammation but more importantly in terms of enhanced organ function.

Example 9

IMP therapy relieves symptoms of inflammatory bowel disease

The intestines represent a unique organ system that is distinct from the brain, peritoneum or heart. The importance of monocytes in Inflammatory Bowel Disease (IBD) has been described (Bain et al, mucosal Immunol,2012 (Bain et al, mucosal immunology, 2012); xu et al, cell Res.18:1220-1229,2008 (Xu et al, cytostudy, volume 18, pages 1220-1229, 2008)), where a model of colitis induced using sodium dextran sulfate (DSS) indicates the chemokines GM CSF as well as Ly6C ^hi Inflammatory monocytes are involved in the pathogenesis of IBD (Xu et al, cell Res.18:1220-1229,2008 (Xu et al, cytostudy, volume 18, pages 1220-1229, 2008)). Ly6C ^hi A reduction in the transport of inflammatory monocytes into the inflamed colon is thought to reduce the severity of the disease. Thus, IMP treatment was studied in animals with DSS-induced colitis (fig. 5P). The symptoms of colitis were significantly reduced in animals treated with PS-IMP compared to vehicle control (fig. 5G). In addition, GR1 with monocyte morphology into inflamed colon ⁺ The number of cells was reduced as determined by immunohistochemistry (fig. 5R-S). In PS-IMP treated animals, this was marked with reduced epithelial cell destruction and significantly increased numbers Epithelial cells of proliferation marker Ki67 were associated (fig. 8F-G). Epithelial cell proliferation is associated with intestinal recovery following DSS challenge, where IMP perfusion is evident in enabling early repair of DSS-mediated lesions.

Example 10

IMP therapy avoids inflammatory lesions in the myocardial infarction model

The effect of 4 days of naked PLGA-IMP treatment was determined using a permanent left anterior descending arterial occlusion model (Yeap et al Methods In Molecular Biology, in Press (Yeap et al, methods of molecular biology, publications)). In vehicle treated animals, occlusion resulted in increased inflammation. IMP treatment significantly reduced the size of inflammatory lesions, thereby alleviating overall cardiac inflammation. In addition, CD68 in the infarct zone was observed ⁺ The number of macrophages was significantly reduced and IMP treatment resulted in CD68 relative to vehicle-treated controls ⁺ Cell/mm ² Is reduced in number. Importantly, the reduced zone was associated with Ly6C in the spleen ^hi A significant increase in the number of inflammatory monocytes correlates with.

To further illustrate the potential of IMP therapy in the myocardial ischemia (LAD occlusion) -reperfusion model, IMP was tested in rodents, where the left anterior descending coronary artery was occluded, followed by release at the subsequent 30 minutes. IMP treatment was started 24 hours after ligation and continued for 4 days. As shown in fig. 10, at 28 days post IR, infarct scar size was reduced by 45% in the treated animals compared to PBS treated animals. In addition, the systolic ejection fraction was increased by 21% in PLG-IMP treated animals compared to control animals.

These data in the previous examples indicate that IMP can be successfully used in a variety of infectious and non-infectious inflammatory disorders to reduce the transport of inflammatory monocytes to the site of inflammation. This results in significantly reduced clinical disease symptoms and may also enable initiation of repair mechanisms that might otherwise be inhibited by the pro-inflammatory environment produced by inflammatory monocytes.

Example 11

Preparation of negatively charged immune-modified particles (IMP)

To poly (ethylene-maleic anhydride) (PEMA) at D ₂ A solution of poly (lactide-co-glycolic acid) (PLG) in Dichloromethane (DCM) (2 mL,20% w/v) was added dropwise to the solution in O (4 mL,1% w/v). The mixture was sonicated on ice at 16 watts for 30 seconds using a VC 30 sonicator (Ultrasonic Processor). The resulting homogenized crude product is then poured into D ₂ O solution (200 mL, containing 0.5% w/v PEMA). The homogenized slurry was stirred overnight at a speed setting of 3.5 using a Bellstir Multi-stir 9 magnetic stirrer (10W for 10s,16W for 30 s) from Bellco Glass company (inc.).

Results

After stirring for three hours, particle size analysis was performed in disposable polystyrene cuvettes using dynamic light scattering.

a.10w,10 s-Z-average= 499.9 nm-pdi=0.23, peak= 634.5nm

b.16w,10 s-Z-average=528.9 nm-pdi=0.227, peak=657.5 nm

c.16w,30 s-Z-average=471.6 nm-pdi=0.228, peak= 580.5nm

d.16w,60 s-Z-average= 491.1 nm-pdi=0.275, peak= 600.8nm

After completion of the reaction, the resulting crude suspension was then purified.

Purification

Fresh D ₂ O and 10 Xsodium bicarbonate buffer were cooled to 4℃overnight. Using a 40 μm cell strainer, 36mL of the particle suspension was filtered from each batch into a suitably labeled 50mL centrifuge tube containing 4mL of cooled 10 Xsodium bicarbonate buffer. Approximately 6 such tubes were made per beaker. All tubes were centrifuged at 7000g for about 15 minutes at 4℃and the supernatant aspirated. The preparation of the suspension was repeated using the procedure described above, with as much of the pellet suspended as possible in 1mL of cooled D ₂ O.

The suspended particles were transferred to a new tube containing 4mL of cooled 10x sodium bicarbonate buffer. (step 1)

The re-suspension of particles is repeated until all particle sediment is successfully re-suspended. (step 2)

The 6 centrifuge tubes were then combined into one centrifuge tube (50 mL tube) and the tube was filled with a remaining volume of cooled D to 40mL ₂ O (washing 1).

The tube was centrifuged at 7000g for 20 minutes at 4℃and the supernatant was aspirated.

Steps 1 and 2 and wash 1 of the resulting particles were repeated at least twice more each time. Finally, the resulting pellet was flash frozen in liquid nitrogen and lyophilized to dryness in a manifold to obtain negatively charged IMP.

Figure 14 shows characterization of surface functionalized poly (lactide-co-glycolide) particles by dynamic light scattering analysis. At 2.5X10 in 18.2M omega water on a Markov laser particle sizer (Malvern Zetasizer Nano ZS) (Markov instruments Inc. (Malvern Instruments, westborough, mass.)) ⁵ Count rate per second surface functionalized poly (lactide-co-glycolide) particles were analyzed. The population of surface functionalized poly (lactide-co-glycolide) particles had a Z-average diameter of 567nm, a peak diameter of 670nm and a polydispersity index of 0.209.

Table 4 shows the measured values of the surface functionalized PLG-PEMA particles. The data in the table are representative, as each batch is slightly different. The numbers in the table are based on a combination of several batches of particles. The measurements of the double emulsified particles are similar to those in table 3.

TABLE 4 measurement of surface-functionalized PLG-PEMA particles

Example 12

IMP-induced regulatory T cells

IMP perfusion in experimental autoimmune encephalomyelitis of MS model has previously been demonstrated to inhibit disease. In this model, on day 0, mice were immunized with Freund's complete adjuvant and myelin antigen, proteolipid protein 139-158. Disease symptoms were monitored daily and when the onset of disease was apparent, IMP treatment was performed for 10 days. In this example, daily intravenous infusion of biodegradable PLGA-IMP lasted 10 days from the onset of disease, not only improved the disease during treatment, but also correlated with asymptomatic 14 days post-treatment time (fig. 15A). The decreased disease score of animals treated at the time of onset was correlated with an increased number of c4+cd25+foxp3+tim3+ regulatory T cells during treatment (fig. 15B and C). On day 20 (10 days after treatment initiation), these tregs expressed negative co-stimulatory molecules PD-1 (fig. 15B). 10 days after cessation of dosing, tregs were found to drop in number to baseline, which was associated with disease recurrence (fig. 15A).

Example 13

IMP binds to a different protein in the inflammatory blood compared to the initial blood

Mouse plasma was collected into heparin tubes from west nile virus infected or mock (uninfected) mice by cardiac puncture. The plasma is then separated from other blood components (e.g., white blood cells and red blood cells) by standard methods well known in the art. Plasma was then combined with 3X 10 ⁸ The immunomodifying nanoparticles were incubated together for 30 minutes at room temperature. After 30 minutes, the particles were washed and proteins were separated on an SDS-PAGE gel, and each lane was cut into 12 equivalent gel slices. The flakes were trypsinized and the resulting peptide mixture was analyzed by reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). GeLC-MS analysis was performed on the proteins. Briefly, proteins were reduced and alkylated in the presence of dithiothreitol and iodoacetamide. The strips were dried by vacuum centrifugation and incubated with 240ng trypsin for 1 hour at 4 ℃. Excess trypsin was removed and replaced with 50mM ammonium bicarbonate for incubation overnight at 37 ℃. Prior to mass spectrometry, peptides were concentrated and desalted using prefabricated microcolumns (foreground biosystems, inc (Perseptive Biosystems, framingham MA) of Framingham, massachusetts) containing Poros R2 resin. The eluted peptide was redissolved in 0.1% formic acid and subjected to reverse phase LC-MS/MS. Approximately 41,000 MS/MS spectra were generated by repeating LC-MS/MS experiments. In MascotTandem mass spectrometry was extracted, the charge state was deconvoluted and the isotope spectrum (deiotonated) was subtracted, and Mascot and X-! The analysis was performed by Tandem. Data were searched for mouse (mouse) entries included in the SWISS-PROT database, with the significance threshold set to p <0.05. The search was performed with a fragment ion mass tolerance of 15ppm and a precursor ion tolerance of 0.2 Da. Methionine sulfoxide, ureido-cysteine, deamidated asparagine and glutamine are designated as variable modifications. Multiple MS/MS experiments were integrated using scaffolds and MS/MS-based peptide and protein identification was validated. These peptides were considered only when the Mascot ion score exceeded 30, 40 and 40 for the doubly, tri-and tetra-charged peptides, respectively. X-! The Tandem identification requires-Log (expected score) scores greater than 2.0. If a minimum of 2 peptides are identified that match the criteria described above, these proteins are accepted for identification. Five proteins were considered identified, although only one peptide was matched. In this case, the peptides were sequenced on multiple occasions, and the single peptide achieved a sequence coverage of greater than 5%.

The results of these studies identified the following proteins that would bind to the particles upon initial serum incubation:

TABLE 5 IMP-binding proteins in initial serum

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Interestingly, however, these methods identified 15 unique proteins that bound to IMP under inflammatory conditions but were not found to bind to IMP under non-inflammatory or initial conditions. These proteins are listed in table 6.

Table 6: proteins that bind only IMP in inflammatory serum

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These results indicate that the particles of the present invention can be used not only as a molecular sink to remove inflammatory mediators, pathological proteins and/or cell debris such as S100 protein and/or fatty acid binding proteins (fig. 16A), but also to concentrate regulatory proteins such as annexin 1 (fig. 16B). In addition, these results can be used in the diagnostic methods described herein, wherein serum is incubated with IMP in vitro or in vivo, and the protein is subsequently purified from IMP by liquid chromatography, mass spectrometry, HPLC or immunoprecipitation, e.g., to determine/diagnose the presence of an inflammatory immune response in a subject (fig. 17).

While specific embodiments of the invention have been described and illustrated, these embodiments should be considered illustrative of the invention only and not as limiting the invention, which is construed in accordance with the accompanying claims.

All patents, applications, and other references cited herein are incorporated by reference in their entirety.

The invention provides:

1. a method of inducing apoptosis of monocytes and/or neutrophils in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles do not contain an attached peptide or antigen moiety.

2. The method of item 1, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

3. The method of item 2, wherein the particles are polystyrene particles.

4. The method of item 2, wherein the particles are diamond particles.

5. The method of item 2, wherein the particles are PLGA particles.

6. The method of item 1, wherein the particles are carboxylated.

7. The method of items 1 and 6, wherein the particles have a zeta potential of less than about-100 mV.

8. The method of item 7, wherein the particles have a zeta potential of between-75 mV and 0 mV.

9. The method of item 8, wherein the particles have a zeta potential of between-50 mV and 0 mV.

10. The method of item 9, wherein the particles have a zeta potential of between-50 mV and-40 mV.

11. The method of item 1, wherein the composition improves inflammatory immune response.

12. The method of item 1, wherein infiltration of inflammatory monocytes, granulocytes or inhibitory neutrophils to the inflammatory lesion is reduced and/or inhibited.

13. The method of clauses 1 and 6, wherein the diameter of the negatively charged particles is between about 0.1 μm and about 10 μm.

14. The method of claim 13, wherein the diameter of the negatively charged particles is between about 0.3 μm and about 5 μm.

15. The method of item 14, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 3 μιη.

16. The method of item 15, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 1 μιη.

17. The method of item 16, wherein the diameter of the negatively charged particles is about 0.5 μm.

18. The method of item 1, wherein the subject has an autoimmune disorder.

19. The method of item 18, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

20. The method of claim 19, wherein the autoimmune disease is multiple sclerosis.

21. The method of item 1, wherein the subject is a transplant recipient.

22. The method of claim 1, wherein the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction.

23. The method of item 1, wherein the subject has psoriasis or dermatitis.

24. The method of item 1, wherein the subject suffers from an allergic disorder.

25. The method of item 24, wherein the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity.

26. The method of item 1, wherein the subject has a bacterial or viral infection.

27. The method of claim 26, wherein the viral infection is a herpes virus infection, a hepatitis virus infection, a west nile virus infection, a flavivirus, an influenza virus infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection.

28. The method of claim 27, wherein the viral infection causes the central nervous system of the subject to be infected.

29. The method of item 28, wherein the viral infection causes viral encephalitis or viral meningitis.

30. The method of claim 26, wherein the bacterial infection infects the central nervous system of said subject.

31. The method of item 30, wherein the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

32. The method of item 1, wherein the subject has received or will receive surgery.

33. The method of item 32, wherein the negatively charged particles are administered to the subject prior to surgery.

34. The method of item 32, wherein the negatively charged particles are administered to the subject during surgery.

35. The method of item 32, wherein the negatively charged particles are administered to the subject after surgery.

36. The method of item 1, wherein the subject has experienced physical trauma or injury.

37. The method of item 1, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

38. A method for removing a pro-inflammatory mediator from the inflammatory environment of a subject having an inflammatory disorder, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles do not contain an attached peptide or antigen moiety.

39. The method of claim 38, wherein the pro-inflammatory mediators produced in the subject bind to the negatively charged particles.

40. The method of item 39, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

41. The method of item 40, wherein the particles are polystyrene particles.

42. The method of item 40, wherein the particles are diamond particles.

43. The method of item 40, wherein the particles are PLGA particles.

44. The method of item 38, wherein the particles are carboxylated.

45. The method of claim 38 or 44, wherein the particles have a zeta potential of less than about-100 mV.

46. The method of item 45, wherein the particles have a zeta potential of between-75 mV and 0 mV.

47. The method of item 46, wherein the particles have a zeta potential of between-50 mV and 0 mV.

48. The method of item 47, wherein the particles have a zeta potential of between-50 mV and-40 mV.

49. The method of item 38, wherein the composition improves inflammatory immune response.

50. The method of claim 38, wherein inflammatory monocytes, monocyte derived suppressor cells and/or inhibitory neutrophils are reduced in infiltration and/or inhibited into the inflammatory lesion.

51. The method of claims 38 and 44, wherein the diameter of the negatively charged particles is between about 0.1 μm and about 10 μm.

52. The method of item 51, wherein the diameter of the negatively charged particles is between about 0.3 μm and about 5 μm.

53. The method of item 52, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 3 μιη.

54. The method of item 53, wherein the diameter of the negatively charged particles is between about 0.5 μm and about 1 μm.

55. The method of item 54, wherein the diameter of the negatively charged particles is about 0.5 μm.

56. The method of claim 38, wherein the subject has an autoimmune disorder.

57. The method of item 56, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

58. The method of item 57, wherein the autoimmune disease is multiple sclerosis.

59. The method of claim 38, wherein the subject is a transplant recipient.

60. The method of claim 38, wherein the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction.

61. The method of item 38, wherein the subject has psoriasis or dermatitis.

62. The method of claim 38, wherein the subject is suffering from an allergic disorder.

63. The method of item 62, wherein the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity.

64. The method of item 38, wherein the subject has a bacterial or viral infection.

65. The method of item 64, wherein the viral infection is a herpes virus infection, a hepatitis virus infection, a west nile virus infection, a flavivirus, an influenza virus infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection.

66. The method of item 65, wherein the viral infection causes the central nervous system of the subject to be infected.

67. The method of item 66, wherein the viral infection causes viral encephalitis or viral meningitis.

68. The method of item 64, wherein the bacterial infection infects the central nervous system of said subject.

69. The method of claim 68, wherein the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

70. The method of item 38, wherein the subject has received or will receive surgery.

71. The method of item 70, wherein the negatively charged particles are administered to the subject prior to surgery.

72. The method of item 70, wherein the negatively charged particles are administered to the subject during surgery.

73. The method of item 70, wherein the negatively charged particles are administered to the subject after surgery.

74. The method of item 38, wherein the subject has experienced physical trauma or injury.

75. The method of claim 38, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

76. A method of inducing antigen-specific tolerance in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles having one or more antigens embedded therein, and a carrier.

77. The method of item 76, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

78. The method of item 77, wherein said particles are polystyrene particles.

79. The method of item 77, wherein said particles are diamond particles.

80. The method of item 77, wherein said particles are PLGA particles.

81. The method of item 76, wherein the particles are carboxylated.

82. The method of claim 76 or 81, wherein the particles have a zeta potential of less than about-100 mV.

83. The method of item 82, wherein the particles have a zeta potential of between-75 mV and +0 mV.

84. The method of item 83, wherein the particles have a zeta potential of between-50 mV and 0 mV.

85. The method of item 84, wherein the particles have a zeta potential of between-50 mV and-40 mV.

86. The method of claim 76, wherein the composition improves inflammatory immune response.

87. The method of claim 76, wherein infiltration of inflammatory monocytes, granulocytes and/or inhibitory neutrophils to the inflammatory lesion is reduced and/or inhibited.

88. The method of claim 76 or 81, wherein the diameter of the negatively charged particles is between about 0.1 μιη to about 10 μιη.

89. The method of claim 88, wherein the diameter of the negatively charged particles is between about 0.3 μm and about 5 μm.

90. The method of claim 89, wherein the diameter of said negatively charged particles is between about 0.5 μm and about 3 μm.

91. The method of item 90, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 1 μιη.

92. The method of item 91, wherein the diameter of the negatively charged particles is about 0.5 μm.

93. The method of claim 76, wherein the subject has an autoimmune disorder.

94. The method of claim 93, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

95. The method of item 94, wherein the autoimmune disease is multiple sclerosis.

96. The method of claim 76, wherein the subject is a transplant recipient.

97. The method of claim 76, wherein the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction.

98. The method of claim 76, wherein the subject has psoriasis or dermatitis.

99. The method of claim 76, wherein the subject is suffering from an allergic disorder.

100. The method of claim 99, wherein the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity.

101. The method of claim 76, wherein the subject has a bacterial or viral infection.

102. The method of item 101, wherein the viral infection is a herpes viral infection, a hepatitis viral infection, a west nile viral infection, a flavivirus, an influenza viral infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection.

103. The method of item 102, wherein the viral infection causes the central nervous system of the subject to be infected.

104. The method of item 103, wherein the viral infection causes viral encephalitis or viral meningitis.

105. The method of item 101, wherein the bacterial infection infects the central nervous system of said subject.

106. The method of item 105, wherein the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

107. The method of item 76, wherein the subject has received or is about to receive surgery.

108. The method of claim 107, wherein the negatively charged particles are administered to the subject prior to surgery.

109. The method of claim 107, wherein the negatively charged particles are administered to the subject during surgery.

110. The method of claim 107, wherein the negatively charged particles are administered to the subject after surgery.

111. The method of item 76, wherein the subject has experienced physical trauma or injury.

112. The method of claim 76, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

113. The method of item 22, 60 or 97, wherein the composition provides protection in the subject from inflammatory injury following myocardial infarction.

114. The method of item 22, 60 or 97, wherein the composition provides protection in the subject from inflammatory injury following reperfusion.

115. A method of enhancing regeneration of damaged tissue in a patient in need thereof, the method comprising administering a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles do not contain an attached peptide or antigen moiety.

116. The method of claim 115, wherein epithelial cell regeneration is enhanced in a patient with colitis.

117. The method of claim 115, wherein remyelination is enhanced in a patient suffering from multiple sclerosis.

118. A method of inducing apoptosis of monocytes, granulocytes and/or neutrophils in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles comprise an antigen comprising one or more epitopes associated with an allergic reaction, an autoimmune disease and/or an inflammatory disease or disorder.

119. A method of inducing antigen-specific tolerance in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles comprise an antigen comprising one or more epitopes associated with an allergy, an autoimmune disease, and/or an inflammatory disease or disorder.

120. The method of item 118 or 119, wherein the one or more epitopes are selected from those described in table 1 or 2.

121. The method of item 118 or 119, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

122. The method of item 121, wherein the particles are carboxylated.

123. The method of claim 118 or 119, wherein the particles have a zeta potential of less than about-100 mV.

124. The method of item 123, wherein the particles have a zeta potential of between-75 mV and +0 mV.

125. The method of item 124, wherein the particles have a zeta potential of between-50 mV and 0 mV.

126. The method of item 125, wherein the particles have a zeta potential of between-50 mV and-40 mV.

127. The method of item 118 or 119, wherein the composition improves an inflammatory immune response.

128. The method of item 118 or 119, wherein infiltration of inflammatory monocytes and/or inhibitory neutrophils to the inflammatory lesion is reduced and/or inhibited.

129. The method of claim 118 or 119, wherein the diameter of the negatively charged particles is between about 0.1 μιη to about 10 μιη.

130. The method of claim 129, wherein the diameter of the negatively charged particles is between about 0.3 μm and about 5 μm.

131. The method of item 130, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 3 μιη.

132. The method of item 131, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 1 μιη.

133. The method of claim 132, wherein the diameter of the negatively charged particles is about 0.5 μm.

134. The method of item 118 or 119, wherein the subject has an autoimmune disorder.

135. The method of claim 134, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, reynolds's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

136. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of myelin basic protein.

137. The composition of item 136, wherein the antigen comprises one or more epitopes of SEQ ID NO. 4975 or SEQ ID NO. 4976.

138. The composition of claim 118 or 119, wherein the antigen comprises one or more epitopes of myelin oligodendrocyte glycoprotein.

139. The composition of item 138, wherein the antigen comprises one or more epitopes of SEQ ID NO. 1 or SEQ ID NO. 4978.

140. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of insulin.

141. The composition of item 140, wherein the antigen comprises one or more epitopes of SEQ ID No. 4981.

142. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of glutamate decarboxylase (GAD).

143. The composition of item 142, wherein the antigen comprises one or more epitopes of SEQ ID No. 4982.

144. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of a proteolipid protein.

145. The composition of item 144, wherein the antigen comprises one or more epitopes of SEQ ID No. 4977.

146. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of gliadin.

147. The composition of item 147, wherein the antigen comprises one or more epitopes of SEQ ID NO:4983-4985 or 5136-5140.

148. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of aquaporin.

149. The composition of item 149, wherein the antigen comprises one or more epitopes of SEQ ID No. 4979.

150. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of myelin-associated glycoprotein.

151. The composition of item 150, wherein the antigen comprises one or more epitopes of SEQ ID No. 4980.

152. The composition of item 118 or 119, wherein the antigen comprises one or more epitopes of the alpha 3 chain of type IV collagen.

153. The composition of item 152, wherein the antigen comprises one or more epitopes of SEQ ID No. 5017.

154. A method for inducing regulatory T cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the negatively charged particles do not contain an attached peptide or antigen moiety.

155. The method of claim 154, wherein the regulatory T cells are CD4 ⁺ T cells.

156. The method of item 155, wherein the CD4 ⁺ Regulatory T cells are CD25 ⁺ And-

Or FoxP3 ⁺ 。

157. The method of claim 154, wherein the regulatory T cells are CD4 ⁺ CD25 ⁺ FoxP3 ⁺ 。

158. The method of item 154, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

159. The method of claim 158, wherein the particles are polystyrene particles.

160. The method of claim 158, wherein the particles are diamond particles.

161. The method of claim 158, wherein the particles are PLGA particles.

162. The method of item 154, wherein the particles are carboxylated.

163. The method of claim 154 or 162, wherein the particles have a zeta potential of less than about-100 mV.

164. The method of item 163, wherein the particles have a zeta potential of between-75 mV and 0 mV.

165. The method of item 164, wherein the particles have a zeta potential of between-50 mV and 0 mV.

166. The method of item 165, wherein the particle has a zeta potential of between-50 mV and-40 mV.

167. The method of claim 154 or 162, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

168. The method of item 167, wherein the diameter of the negatively charged particles is between about 0.3 μιη to about 5 μιη.

169. The method of claim 168, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 3 μιη.

170. The method of claim 169, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 1 μιη.

171. The method of claim 170, wherein the diameter of the negatively charged particles is about 0.5 μm.

172. The method of claim 154, wherein the subject has an autoimmune disorder.

173. The method of claim 172, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, reynolds's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

174. The method of item 173, wherein the autoimmune disease is multiple sclerosis.

175. The method of claim 154, wherein the subject is a transplant recipient.

176. The method of claim 154, wherein the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction.

177. The method of claim 154, wherein the subject has psoriasis or dermatitis.

178. The method of claim 154, wherein the subject is suffering from an allergic disorder.

179. The method of claim 178, wherein the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity.

180. The method of claim 154, wherein the subject has a bacterial or viral infection.

181. The method of item 180, wherein the viral infection is a herpes viral infection, a hepatitis viral infection, a west nile viral infection, a flavivirus, an influenza viral infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection.

182. The method of item 181, wherein the viral infection causes the central nervous system of the subject to be infected.

183. The method of item 182, wherein the viral infection causes viral encephalitis or viral meningitis.

184. The method of item 180, wherein the bacterial infection infects the central nervous system of said subject.

185. The method of item 184, wherein the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

186. The method of claim 154, wherein the subject has received or will receive surgery.

187. The method of claim 186, wherein the negatively charged particles are administered to the subject prior to surgery.

188. The method of claim 186, wherein the negatively charged particles are administered to the subject during surgery.

189. The method of claim 186, wherein the negatively charged particles are administered to the subject after surgery.

190. The method of item 154, wherein the subject has experienced physical trauma or injury.

191. The method of claim 154, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

192. A method for concentrating a regulatory protein from serum or plasma of a subject having an inflammatory disorder, the method comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a carrier, wherein the particles do not contain an attached peptide or antigen moiety.

193. The method of claim 38, wherein the regulatory protein produced in the subject binds to the negatively charged particles.

194. The method of item 193, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

195. The method of item 194, wherein the particles are polystyrene particles.

196. The method of claim 194, wherein the particles are diamond particles.

197. The method of item 194, wherein the particles are PLGA particles.

198. The method of item 192, wherein the particles are carboxylated.

199. The method of item 192 or 198, wherein the particles have a zeta potential of less than about-100 mV.

200. The method of claim 199, wherein the particles have a zeta potential of between-75 mV and 0 mV.

201. The method of item 200, wherein the particles have a zeta potential of between-50 mV and 0 mV.

202. The method of item 201, wherein the particles have a zeta potential of between-50 mV and-40 mV.

203. The method of claim 192, wherein the composition improves inflammatory immune response.

204. The method of claim 192, wherein inflammatory monocytes, monocyte derived suppressor cells and/or inhibitory neutrophils are reduced in infiltration and/or inhibited into the inflammatory focus.

205. The method of items 192 and 198, wherein the diameter of the negatively charged particles is between about 0.1 μιη to about 10 μιη.

206. The method of claim 205, wherein the diameter of said negatively charged particles is between about 0.3 μm and about 5 μm.

207. The method of item 206, wherein the diameter of the negatively charged particles is between about 0.5 μιη to about 3 μιη.

208. The method of claim 207, wherein the diameter of said negatively charged particles is between about 0.5 μm and about 1 μm.

209. The method of claim 208, wherein the diameter of said negatively charged particles is about 0.5 μm.

210. The method of claim 192, wherein the subject has an autoimmune disorder.

211. The method of item 210, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, raynaud's syndrome, sjogren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic Lateral Sclerosis (ALS), celiac disease, ulcerative colitis, and crohn's disease.

212. The method of item 211, wherein the autoimmune disease is multiple sclerosis.

213. The method of claim 192, wherein the subject is a transplant recipient.

214. The method of claim 192, wherein the subject has ischemia reperfusion injury, atherosclerosis or is suffering from myocardial infarction.

215. The method of claim 192, wherein the subject has psoriasis or dermatitis.

216. The method of claim 192, wherein the subject is suffering from an allergic disorder.

217. The method of claim 216, wherein the allergic disorder is eczema, asthma, allergic rhinitis, or skin hypersensitivity.

218. The method of claim 192, wherein the subject has a bacterial or viral infection.

219. The method of claim 218, wherein the viral infection is a herpes viral infection, a hepatitis viral infection, a west nile viral infection, a flavivirus, an influenza viral infection, a rhinovirus infection, a papillomavirus infection, or a parainfluenza virus infection.

220. The method of item 219, wherein the viral infection causes the central nervous system of the subject to be infected.

221. The method of item 220, wherein the viral infection causes viral encephalitis or viral meningitis.

222. The method of claim 218, wherein the bacterial infection infects the central nervous system of said subject.

223. The method of claim 68, wherein the bacterial infection causes sepsis bacterial encephalitis or bacterial meningitis.

224. The method of claim 192, wherein the subject has received or is about to receive surgery.

225. The method of item 224, wherein the negatively charged particles are administered to the subject prior to surgery.

226. The method of item 224, wherein the negatively charged particles are administered to the subject during surgery.

227. The method of item 224, wherein the negatively charged particles are administered to the subject after surgery.

228. The method of item 192, wherein the subject has experienced physical trauma or injury.

229. The method of claim 192, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

230. A method for diagnosing an inflammatory state in a subject, the method comprising:

(a) Withdrawing blood from the subject;

(b) Separating the blood to separate serum and/or plasma;

(c) Incubating the serum and/or plasma with negatively charged particles for a period of time;

(d) Isolating or purifying proteins bound to the negatively charged particles; and

(e) Determining that the subject is in an inflammatory state when the protein purified from the negatively charged particles in (d) comprises one or more of the proteins listed in table 6.

231. A method for diagnosing an inflammatory state in a subject, the method comprising:

(a) Administering intravenously to the subject a composition comprising negatively charged particles, wherein

The negatively charged particles do not contain attached peptide or antigen moieties;

(b) Withdrawing the negatively charged particles administered in step (a) from the blood of the subject;

(c) Isolating or purifying proteins bound to the negatively charged particles; and

(d) Determining that the subject is in an inflammatory state when the protein purified from the negatively charged particles in (c) comprises one or more of the proteins listed in table 6.

232. The method of item 230 or 231, wherein the proteins purified from the negatively charged particles in (c) comprise two or more of the proteins listed in table 6.

233. The method of item 230 or 231, wherein the proteins purified from the negatively charged particles in (c) comprise three or more of the proteins listed in table 6.

234. The method of item 230 or 231, wherein the proteins purified from the negatively charged particles in (c) comprise four or more of the proteins listed in table 6.

235. The method of item 230 or 231, wherein the proteins purified from the negatively charged particles in (c) comprise five or more of the proteins listed in table 6.

236. The method of item 230 or 231, wherein the protein purified from the negatively charged particles in (c) comprises ten or more of the proteins listed in table 6.

237. The method of item 230 or 231, wherein the negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly (lactic-co-glycolic acid) (PLGA) particles.

238. The method of item 237, wherein the particles are polystyrene particles.

239. The method of claim 237, wherein the particles are diamond particles.

240. The method of item 237, wherein the particles are PLGA particles.

241. The method of item 230 or 231, wherein the particle is carboxylated.

242. The method of item 241, wherein the particles have a zeta potential of less than about-100 mV.

243. The method of item 242, wherein the particles have a zeta potential of between-75 mV and 0 mV.

244. The method of claim 243, wherein said particles have a zeta potential of between-50 mV and 0 mV.

245. The method of claim 244, wherein the particles have a zeta potential of between-50 mV and-40 mV.

246. The method of item 230 or 231, wherein the diameter of the negatively charged particles is between about 0.1 μιη to about 10 μιη.

247. The method of claim 246, wherein the diameter of said negatively charged particles is between about 0.3 μm and about 5 μm.

248. The method of claim 247, wherein the diameter of the negatively charged particles is between about 0.5 μm and about 3 μm.

249. The method of item 248, wherein the diameter of said negatively charged particles is between about 0.5 μm and about 1 μm.

250. The method of item 249, wherein the diameter of the negatively charged particles is about 0.5 μm.

Claims

1. Use of a pharmaceutical composition comprising negatively charged PLGA, PLA, or PGA particles, said particles having an average zeta potential of between-75 mV and-30 mV, and a carrier, wherein said negatively charged particles do not contain other therapeutic agents, in the manufacture of a composition for treating a subject suffering from ischemia-reperfusion injury.

2. The use of claim 1, wherein the negatively charged particles are PLGA particles.

3. The use of claim 1, wherein the negatively charged particles are carboxylated.

4. The use of claim 1, wherein the negatively charged particles have an average zeta potential of between-50 mV and-30 mV.

5. The use of claim 1, wherein the diameter of the negatively charged particles is between 0.1 μm and 10 μm.

6. The use according to claim 5, wherein the diameter of the negatively charged particles is between 0.3 μm and 5 μm.

7. The use of claim 6, wherein the diameter of the negatively charged particles is between 0.3 μm and 3 μm.

8. The use of claim 7, wherein the diameter of the negatively charged particles is between 0.3 μm and 1 μm.

9. The use of claim 8, wherein the diameter of the negatively charged particles is 0.5 μm.

10. The use of claim 1, wherein the subject has undergone or will undergo surgery.

11. The use of claim 10, wherein the negatively charged particles are administered to the subject prior to surgery.

12. The use of claim 11, wherein the negatively charged particles are administered to the subject during surgery.

13. The use of claim 11, wherein the negatively charged particles are administered to the subject after surgery.

14. The use of claim 1, wherein the subject has experienced injury.

15. The use of claim 14, wherein the injury is a physical wound.

16. The use of claim 1, wherein the pharmaceutical composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally or subcutaneously.

17. The use of claim 1, wherein the pharmaceutical composition is administered intravenously.